Genetic Diversity of White Sapote (Casimiroa edulis La Llave & Lex.) Demonstrated by Intersimple Sequence Repeat Analysis

Authors:
Masashi Yamamoto Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Takahiro Tomita Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Michio Onjo Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Kiyotake Ishihata Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Tatsuya Kubo Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Shigeto Tominaga Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Yoshimi Yonemoto Tropical Agriculture Research Front, Japan International Research Centre for Agricultural Sciences, Ishigaki 907-0002, Japan

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Abstract

Phylogenic relationships among 31 cultivars of white sapote (Casimiroa edulis La Llave & Lex.) were examined by intersimple sequence repeat (ISSR) analysis. Polymorphic fragments were obtained in 24 of 100 ISSR primers (UBC Set#9, 801–900). Although ‘Cuccio’ and ‘Florida’ showed identical fragment patterns, the remaining cultivars could be distinguished from each other. The present study demonstrated the usefulness of ISSR analysis for cultivar identification and phylogenic study in white sapote.

White sapote (Casimiroa edulis La Llave & Lex.) is an evergreen fruit tree that originated in the highlands of Mexico and Central America (Morton, 1987; Yonemoto et al., 2007). Because white sapote adapts to subtropical climate and produces attractive, relatively large and very sweet fruit, it is grown not only in the United States and Mexico, but also in warm regions of New Zealand, Australia, Israel, and Japan.

A large number of cultivars exist in white sapote because superior clones or chance seedlings were selected in California and Florida (Morton, 1987). However, the parentage of many cultivars is unknown (Brooks and Olmo, 1972), and their genetic relationships remain unclear. Recently, floral morphology, randomly amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) markers (Yonemoto et al., 2007) have been used to characterize the white sapote germplasm.

In a previous study (Yamamoto et al., 2006), we conducted isozyme analysis for cultivar identification of white sapote. Cultivars could be classified into groups, but it was impossible to distinguish cultivars within a group from each other.

DNA analysis has been a powerful tool for cultivar identification and related studies. Intersimple sequence repeat (ISSR) analysis is a simple and quick method and its markers are highly polymorphic (Pradeep Reddy et al., 2002). In fruit trees as well, this technique has been used to elucidate genetic diversity and for phylogenic studies of citrus (Fang and Roose, 1997; Fang et al., 1997) and grapes (Moreno et al., 1998).

In the present study, we examined ISSR analysis to identify each cultivar and clarify phylogenic relationships of white sapote using our germplasm collection.

Materials and Methods

Plant material and DNA extraction.

White sapote cultivars preserved at the Ibusuki Experimental Botanical Garden of the Experimental Farm, Faculty of Agriculture, Kagoshima University (Ibusuki, Kagoshima Prefecture, Japan) were used. In this study, we analyzed 31 cultivars (Table 1). Total DNA was extracted from leaves according to the SDS method of Honda and Hirai (1990).

Table 1.

White sapote cultivars used in this study and their types of flowers.

Table 1.

Intersimple sequence repeat analysis.

Primers (UBC Set#9, 801–900) were purchased from the University of British Columbia. Polymerase chain reactions were performed in a Gene Amp9700 (Applied Biosystems, Foster, CA) thermal cycler programmed as follows: initial heating at 94 °C for 5 min, 40 cycles of denaturing at 94 °C for 1 min, annealing at 52 °C for 45 s, extension at 72 °C for 2 min, and a final extension of 10 min at 72 °C. Amplified products were electrophoresed on 1.5% agarose gels and detected by staining with Mupid-Blue (Advancebio, Tokyo). The bands were recorded as 1 for present and as 0 for absent. Genetic distance was calculated between each pair of the cultivars (Nei and Li, 1979). For phylogenic analysis, a tree was constructed with Molecular Evolutionary Genetic Analysis (MEGA, ver. 3.1, The Biodesign Institute, Tempe, AZ) software (Kumar et al., 2004) by applying the unweighted pair group method with arithmetic averages.

Results and Discussion

The sizes of almost all amplified fragments ranged from 500 to 2000 base pairs. Twenty-four of 100 primers showed a clear polymorphic fragment (Fig. 1; Table 2). There were 49 polymorphic fragments in total and 2.0 polymorphic fragments per primer on average. The number of polymorphic fragments produced by each primer ranged from one by #821, #828, #844, #846, #848, #856, and #857 to five by #812.

Fig. 1.
Fig. 1.

DNA amplifications of white sapote cultivars using intersimple sequence repeat primer #881. 1 = Rixford; 2 = Salad; 3 = Selk; 4 = Vernon; 5 = Vista; 6 = Walton; 7 = White; 8 = Yellow. M = molecular markers.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1329

Table 2.

List of primers and their sequence demonstrating polymorphic fragment in this study.

Table 2.

The dendrogram was constructed from ISSR data (Fig. 2). All cultivars except for ‘Cuccio’ and ‘Florida’ could be distinguished from each other. ‘Cuccio’ and ‘Florida’ always showed identical fragment patterns. Major cultivars for fruit production such as ‘Cuccio’ and ‘Pike’ clustered together. ‘McDill’ and ‘Walton’ were distant from the others.

Fig. 2.
Fig. 2.

Dendrogram of 31 white sapote cultivars generated by unweighted pair group method with arithmetic averages cluster analysis of intersimple sequence repeat data.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1329

The present study demonstrated the usefulness of ISSR analysis for cultivar identification and phylogenic study in white sapote, although ‘Cuccio’ and ‘Florida’ could not be distinguished. Both cultivars have been considered identical according to morphological traits (California rare fruit growers, 1996). The present findings on ISSR analysis support this concept, although Yonemoto et al. (2007) reported that they are distinct cultivars showing close genetic relation between them using RAPD and AFLP markers.

Some cultivars such as ‘Max Golden’, which has hairy leaves and is called woolly leaved white sapote, has been considered a distinct species C. tetrameria Millsp. (Brooks and Olmo, 1972). However, ‘Max Golden’ was not distinct from others and clustered with ‘Cuccio’ and ‘Pike’ in our ISSR data. Such type of cluster was reported by Yonemoto et al. (2007) using an RAPD marker. This finding agrees with Morton (1987) who considered that woolly leaved white sapote may only be a variant of C. edulis.

There are three types of flowers in white sapote: type I, with a large ovary and lacking pollen; type II, with a small ovary and producing pollen; and type III, with a large ovary and producing pollen (Chambers, 1984; Yonemoto et al., 2001, 2007). In general, type I cultivars bear heavily and produce large fruit, whereas the productivity of type II cultivars is unstable (Chambers, 1984; Yonemoto et al., 2001). Table 1 shows the flower type of cultivars used in this study according to Yonemoto et al. (2001). In the cluster including ‘Cuccio’ and ‘Max Golden’, all cultivars have type I flower except for type III ‘Maltby’. This indicates a close relationship among those type I cultivars. However, flower types do not correspond to the clusters formed by the remaining cultivars.

The present study demonstrated the efficiency of ISSR analysis for cultivar identification of white sapote. Moreover, ISSR analysis could provide considerable information about genetic relationships among the cultivars. These data are considered useful information for future genetic improvement of white sapote.

Literature Cited

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  • Chambers, R.R. 1984 White sapote varieties progress report California Rare Fruit Growers Yrbk. 16 56 64

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  • Fang, D.Q. , Roose, M.L. , Kruegar, R.R. & Federici, C.T. 1997 Fingerprinting trifoliate orange germplasm accessions with isozymes, RFLPs and inter-simple sequence repeat markers Theor. Appl. Genet. 95 211 219

    • Search Google Scholar
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  • Honda, H. & Hirai, A. 1990 A simple and efficient method for identification of hybrids using nonradioactive rDNA as probe Jpn. J. Breeding 40 339 348

    • Search Google Scholar
    • Export Citation
  • Kumar, S. , Tamura, K. & Nei, M. 2004 MEGA 3: Integrated software for molecular evolutionary genetic analysis and sequence alignment Brief Bioinformatics 5 150 163

    • Search Google Scholar
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  • Moreno, S. , Martin, J.P. & Oltiz, J.M. 1998 Inter-simple sequence repeat PCR for characterization of closely related grapevine germplasm Euphytica 101 117 125

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  • Morton, J.F. 1987 White sapote 191 196 Fruits of warm climate Creative Resource System Winterville, NC

  • Nei, M. & Li, W.H. 1979 Mathematical model for studying genetic variation in terms of restriction endonucleases Proc. Natl. Acad. Sci. USA 76 5269 5273

    • Search Google Scholar
    • Export Citation
  • Pradeep Reddy, M. , Sarla, N. & Siddiq, E.A. 2002 Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding Euphytica 128 9 17

    • Search Google Scholar
    • Export Citation
  • Yamamoto, M. , Nakagawa, T. , Onjo, M. , Yonemoto, Y. , Ishihata, K. , Kubo, T. & Tominaga, S. 2006 Isozyme analysis of white sapote (Casimiroa edulis La Llave & Lex) Bul. Fac. Agr. Kagoshima Univ. 56 15 18

    • Search Google Scholar
    • Export Citation
  • Yonemoto, Y. , Chowdhury, A.K. , Kato, H. , Macha, M.M. & Okuda, H. 2007 Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology, RAPD and AFLP markers Scientia Hort. 112 366 375

    • Search Google Scholar
    • Export Citation
  • Yonemoto, Y. , Higuchi, H. , Ishihata, K. , Ikeda, M. & Tomita, E. 2001 Analysis of varietal differences in floral and fruit morphology in white sapote (Casimiroa edulis Llave and Lex.) Jpn. J. Trop. Agr. 45 38 44

    • Search Google Scholar
    • Export Citation
  • DNA amplifications of white sapote cultivars using intersimple sequence repeat primer #881. 1 = Rixford; 2 = Salad; 3 = Selk; 4 = Vernon; 5 = Vista; 6 = Walton; 7 = White; 8 = Yellow. M = molecular markers.

  • Dendrogram of 31 white sapote cultivars generated by unweighted pair group method with arithmetic averages cluster analysis of intersimple sequence repeat data.

  • Brooks, R.M. & Olmo, H.P. 1972 Register of new fruit and nut varieties 2nd ed Univ. of California Press Berkeley

  • California rare fruit growers 1996 White sapote 5 Feb. 2003 <http//www.crfg.org/pubs/ff/whitesapote.html>.

  • Chambers, R.R. 1984 White sapote varieties progress report California Rare Fruit Growers Yrbk. 16 56 64

  • Fang, D.Q. & Roose, M.L. 1997 Identification of closely related citrus cultivars with inter-simple sequence repeat markers Theor. Appl. Genet. 95 408 417

    • Search Google Scholar
    • Export Citation
  • Fang, D.Q. , Roose, M.L. , Kruegar, R.R. & Federici, C.T. 1997 Fingerprinting trifoliate orange germplasm accessions with isozymes, RFLPs and inter-simple sequence repeat markers Theor. Appl. Genet. 95 211 219

    • Search Google Scholar
    • Export Citation
  • Honda, H. & Hirai, A. 1990 A simple and efficient method for identification of hybrids using nonradioactive rDNA as probe Jpn. J. Breeding 40 339 348

    • Search Google Scholar
    • Export Citation
  • Kumar, S. , Tamura, K. & Nei, M. 2004 MEGA 3: Integrated software for molecular evolutionary genetic analysis and sequence alignment Brief Bioinformatics 5 150 163

    • Search Google Scholar
    • Export Citation
  • Moreno, S. , Martin, J.P. & Oltiz, J.M. 1998 Inter-simple sequence repeat PCR for characterization of closely related grapevine germplasm Euphytica 101 117 125

    • Search Google Scholar
    • Export Citation
  • Morton, J.F. 1987 White sapote 191 196 Fruits of warm climate Creative Resource System Winterville, NC

  • Nei, M. & Li, W.H. 1979 Mathematical model for studying genetic variation in terms of restriction endonucleases Proc. Natl. Acad. Sci. USA 76 5269 5273

    • Search Google Scholar
    • Export Citation
  • Pradeep Reddy, M. , Sarla, N. & Siddiq, E.A. 2002 Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding Euphytica 128 9 17

    • Search Google Scholar
    • Export Citation
  • Yamamoto, M. , Nakagawa, T. , Onjo, M. , Yonemoto, Y. , Ishihata, K. , Kubo, T. & Tominaga, S. 2006 Isozyme analysis of white sapote (Casimiroa edulis La Llave & Lex) Bul. Fac. Agr. Kagoshima Univ. 56 15 18

    • Search Google Scholar
    • Export Citation
  • Yonemoto, Y. , Chowdhury, A.K. , Kato, H. , Macha, M.M. & Okuda, H. 2007 Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology, RAPD and AFLP markers Scientia Hort. 112 366 375

    • Search Google Scholar
    • Export Citation
  • Yonemoto, Y. , Higuchi, H. , Ishihata, K. , Ikeda, M. & Tomita, E. 2001 Analysis of varietal differences in floral and fruit morphology in white sapote (Casimiroa edulis Llave and Lex.) Jpn. J. Trop. Agr. 45 38 44

    • Search Google Scholar
    • Export Citation
Masashi Yamamoto Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Takahiro Tomita Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Michio Onjo Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Kiyotake Ishihata Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Tatsuya Kubo Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Shigeto Tominaga Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

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Yoshimi Yonemoto Tropical Agriculture Research Front, Japan International Research Centre for Agricultural Sciences, Ishigaki 907-0002, Japan

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Contributor Notes

To whom reprint requests should be addressed; e-mail yamasa@agri.kagoshima-u.ac.jp.

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  • DNA amplifications of white sapote cultivars using intersimple sequence repeat primer #881. 1 = Rixford; 2 = Salad; 3 = Selk; 4 = Vernon; 5 = Vista; 6 = Walton; 7 = White; 8 = Yellow. M = molecular markers.

  • Dendrogram of 31 white sapote cultivars generated by unweighted pair group method with arithmetic averages cluster analysis of intersimple sequence repeat data.

 

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