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Genetic Diversity of White Sapote (Casimiroa Edulis La Llave & Lex

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Genetic Diversity of White Sapote (Casimiroa Edulis La Llave & Lex HORTSCIENCE 42(6):1329–1331. 2007. was calculated between each pair of the cultivars (Nei and Li, 1979). For phylogenic analysis, a tree was constructed with Molec- Genetic Diversity of White Sapote ular Evolutionary Genetic Analysis (MEGA, ver. 3.1, The Biodesign Institute, Tempe, AZ) (Casimiroa edulis La Llave & Lex.) software (Kumar et al., 2004) by applying the unweighted pair group method with arith- Demonstrated by Intersimple metic averages. Sequence Repeat Analysis Results and Discussion Masashi Yamamoto1, Takahiro Tomita, Michio Onjo, Kiyotake Ishihata, Tatsuya Kubo, and Shigeto Tominaga The sizes of almost all amplified frag- ments ranged from 500 to 2000 base pairs. Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima Twenty-four of 100 primers showed a clear 890-0065, Japan polymorphic fragment (Fig. 1; Table 2). There were 49 polymorphic fragments in Yoshimi Yonemoto total and 2.0 polymorphic fragments per Tropical Agriculture Research Front, Japan International Research Centre primer on average. The number of polymor- for Agricultural Sciences, Ishigaki 907-0002, Japan phic fragments produced by each primer ranged from one by #821, #828, #844, Additional index words. Casimiroa tetrameria, cultivar identification, DNA analysis, #846, #848, #856, and #857 to five by #812. intersimple sequence repeat, phylogenic relationships The dendrogram was constructed from Abstract. Phylogenic relationships among 31 cultivars of white sapote (Casimiroa edulis ISSR data (Fig. 2). All cultivars except for La Llave & Lex.) were examined by intersimple sequence repeat (ISSR) analysis. ‘Cuccio’ and ‘Florida’ could be distinguished Polymorphic fragments were obtained in 24 of 100 ISSR primers (UBC Set#9, 801–900). from each other. ‘Cuccio’ and ‘Florida’ Although ‘Cuccio’ and ‘Florida’ showed identical fragment patterns, the remaining always showed identical fragment patterns. cultivars could be distinguished from each other. The present study demonstrated the Major cultivars for fruit production such as usefulness of ISSR analysis for cultivar identification and phylogenic study in white ‘Cuccio’ and ‘Pike’ clustered together. sapote. ‘McDill’ and ‘Walton’ were distant from the others. The present study demonstrated the Whitesapote(Casimiroa edulis La are highly polymorphic (Pradeep Reddy usefulness of ISSR analysis for cultivar Llave & Lex.) is an evergreen fruit tree et al., 2002). In fruit trees as well, this that originated in the highlands of Mexico technique has been used to elucidate genetic and Central America (Morton, 1987; diversity and for phylogenic studies of citrus Yonemoto et al., 2007). Because white (Fang and Roose, 1997; Fang et al., 1997) and Table 1. White sapote cultivars used in this study sapote adapts to subtropical climate and grapes (Moreno et al., 1998). and their types of flowers. produces attractive, relatively large and In the present study, we examined ISSR Cultivar Flower typez very sweet fruit, it is grown not only in analysis to identify each cultivar and clarify Cate I the United States and Mexico, but also in phylogenic relationships of white sapote Charles Early I warm regions of New Zealand, Australia, using our germplasm collection. Chestnut I Israel, and Japan. Cuccio I A large number of cultivars exist in white Edgehill II sapote because superior clones or chance Materials and Methods Fallbrook II seedlings were selected in California and Florida I Florida (Morton, 1987). However, the par- Plant material and DNA extraction. Fournoy II Golden Globe II entage of many cultivars is unknown (Brooks White sapote cultivars preserved at the Ibu- Gwin I and Olmo, 1972), and their genetic relation- suki Experimental Botanical Garden of the Lammertz I ships remain unclear. Recently, floral mor- Experimental Farm, Faculty of Agriculture, Lamsey Large I phology, randomly amplified polymorphic Kagoshima University (Ibusuki, Kagoshima Malibue No. 1 I DNA (RAPD), and amplified fragment length Prefecture, Japan) were used. In this study, Malibue No. 3 I polymorphism (AFLP) markers (Yonemoto we analyzed 31 cultivars (Table 1). Total Maltby III et al., 2007) have been used to characterize DNA was extracted from leaves according Max Golden I the white sapote germplasm. to the SDS method of Honda and Hirai McDill I In a previous study (Yamamoto et al., (1990). Mexico I Michele II 2006), we conducted isozyme analysis for Intersimple sequence repeat analysis. Pri- Nies I cultivar identification of white sapote. Culti- mers (UBC Set#9, 801–900) were purchased Ortega II vars could be classified into groups, but it was from the University of British Columbia. Pike I impossible to distinguish cultivars within a Polymerase chain reactions were performed Rainbow I group from each other. in a Gene Amp9700 (Applied Biosystems, Rixford II DNA analysis has been a powerful tool for Foster, CA) thermal cycler programmed as Salad I cultivar identification and related studies. follows: initial heating at 94 °C for 5 min, 40 Selk II Intersimple sequence repeat (ISSR) analysis cycles of denaturing at 94 °C for 1 min, Vernon II is a simple and quick method and its markers annealing at 52 °C for 45 s, extension at 72 °C Vista II Walton II for 2 min, and a final extension of 10 min at White I 72 °C. Amplified products were electrophor- Yellow I Received for publication 12 Feb. 2007. Accepted esed on 1.5% agarose gels and detected by zAccording to Yonemoto et al. (2001). Type I = for publication 15 May 2007. staining with Mupid-Blue (Advancebio, large ovary and lacking pollen; type II = small 1To whom reprint requests should be addressed; Tokyo). The bands were recorded as 1 for ovary and producing pollen; type III = large ovary e-mail [email protected]. present and as 0 for absent. Genetic distance and producing pollen. HORTSCIENCE VOL. 42(6) OCTOBER 2007 1329 Fig. 1. DNA amplifications of white sapote culti- vars 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. Table 2. List of primers and their sequence demonstrating polymorphic fragment in this study. Primer numberz Sequencey 808 AGA GAG AGA GAG AGA GC 812 GAG AGA GAG AGA GAG AA 818 CAC ACA CAC ACA CAC AG 820 GTG TGT GTG TGT GTG TC 821 GTG TGT GTG TGT GTG TT 823 TCT CTC TCT CTC TCT CC 825 ACA CAC ACA CAC ACA CT 826 ACA CAC ACA CAC ACA CC 827 ACA CAC ACA CAC ACA CG 828 TGT GTG TGT GTG TGT GA 834 AGA GAG AGA GAG AGA GYT 840 GAG AGA GAG AGA GAG AYT Fig. 2. Dendrogram of 31 white sapote cultivars generated by unweighted pair group method with 844 CTC TCT CTC TCT CTC TRC arithmetic averages cluster analysis of intersimple sequence repeat data. 846 CAC ACA CAC ACA CAC ART 847 CAC ACA CAC ACA CAC ARC 848 CAC ACA CAC ACA CAC ARG 856 ACA CAC ACA CAC ACA CYA with ‘Cuccio’ and ‘Pike’ in our ISSR data. tion about genetic relationships among the 857 ACA CAC ACA CAC ACA CYG Such type of cluster was reported by Yonemoto cultivars. These data are considered useful 859 TGT GTG TGT GTG TGT GRC et al. (2007) using an RAPD marker. This information for future genetic improvement 868 GAA GAA GAA GAA GAA GAA finding agrees with Morton (1987) who of white sapote. 873 GAC AGA CAG ACA GAC A 881 GGG TGG GGT GGG GTG considered that woolly leaved white sapote 886 VDV CTC TCT CTC TCT CT may only be a variant of C. edulis. 895 AGA GTT GGT AGC TCT TGA TC There are three types of flowers in white Literature Cited zUBC Set#9, 801–900. sapote: type I, with a large ovary and lacking Brooks, R.M. and H.P. Olmo. 1972. Register of yY = (C, T); R = (A, G); V = (A, C, G). pollen; type II, with a small ovary and pro- new fruit and nut varieties. 2nd ed Univ. of ducing pollen; and type III, with a large ovary California Press, Berkeley. and producing pollen (Chambers, 1984; California rare fruit growers. 1996. White sapote. Yonemoto et al., 2001, 2007). In general, 5 Feb. 2003. <http//www.crfg.org/pubs/ff/ identification and phylogenic study in white type I cultivars bear heavily and produce whitesapote.html>. sapote, although ‘Cuccio’ and ‘Florida’ could large fruit, whereas the productivity of Chambers, R.R. 1984. White sapote varieties pro- not be distinguished. Both cultivars have type II cultivars is unstable (Chambers, gress report. California Rare Fruit Growers Yrbk. 16:56–64. been considered identical according to mor- 1984; Yonemoto et al., 2001). Table 1 shows Fang, D.Q. and M.L. Roose. 1997. Identification phological traits (California rare fruit the flower type of cultivars used in this of closely related citrus cultivars with inter- growers, 1996). The present findings on ISSR study according to Yonemoto et al. (2001). simple sequence repeat markers. Theor. Appl. analysis support this concept, although Yone- In the cluster including ‘Cuccio’ and ‘Max Genet. 95:408–417. moto et al. (2007) reported that they are Golden’, all cultivars have type I flower Fang, D.Q., M.L. Roose, R.R. Kruegar, and C.T. distinct cultivars showing close genetic rela- except for type III ‘Maltby’. This indicates a Federici. 1997. Fingerprinting trifoliate orange tion between them using RAPD and AFLP close relationship among those type I culti- germplasm accessions with isozymes, RFLPs markers. vars. However, flower types do not corre- and inter-simple sequence repeat markers. Some cultivars such as ‘Max Golden’, spond to the clusters formed by the remaining Theor. Appl. Genet. 95:211–219. Honda, H. and A. Hirai. 1990. A simple and which has hairy leaves and is called woolly cultivars. efficient method for identification of hybrids leaved white sapote, has been considered a The present study demonstrated the effi- using nonradioactive rDNA as probe.
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