Identification of Satsuma Mandarin (Citrus unshiu) Cultivars in California Using Amplified Fragment Length Polymorphism (AFLP) Markers C.-C.T. Chao, P.S. Devanand and B.R. Cao Department of Botany and Plant Sciences University of California Riverside, California 92521 USA Keywords: Citrus aurantium, Citrus reticulata, Citrus vulgaris, Citrus yatsushiro, genetic similarity, diversity, variability Abstract Satsuma mandarin (Citrus unshiu) is one of the most important types of citrus grown world-wide, especially in China and Japan. It is also produced in California from mid-october to January. Several new Satsuma mandarin cultivars, originally from Japan or China, were introduced into California in the past few years. We intend to identify different Satsuma mandarin cultivars in California using Amplified Fragment Length Polymorphism (AFLP) markers. Fourteen Japanese Satsuma mandarin cultivars or selections, four Chinese Satsuma mandarin cultivars, and three Satuma mandarin cultivars of unknown origin were included in the studies. Citrus yatsushiro, C. reticulata, C. vulgaris, and C. aurantium were used as out-group controls. Six primer sets, E+GG/M+CTA, E+CC/M+CTA, E+GC/M+CTC, E+CA/M+CTC, E+TT/M+CTG, and C+AG/M+CTG were used in the AFLP analyses. We were able to differentiate 19 individual Satsuma mandarins. Twenty Satsuma mandarin cultivars were separated into five subgroups based on the unweighted pair-group method using arithmetic average (UPGMA) analysis. The AFLP marker system developed here will be useful for Satsuma mandarin fingerprinting and future Satsuma mandarin germplasm collection and preservation. INTRODUCTION Satsuma mandarins (Citrus unshiu Marcow.) are a group of seedless citrus with high cold tolerance and excellent taste. They are the major type of citrus grown in China and Japan and are also grown in Argentina, New Zealand, South Africa, South Korea, Spain, Turkey, the United States, and Uruguay. In California, the major Satsuma mandarin production areas are in the San Joaquin Valley and the upper Sacramento Valley. The major leading Satsuma mandarin cultivar in California is the Frost Nucellar #1 Owari Satsuma mandarin. There is also some production of Okitsu Wase and Dobashi Beni Satsuma mandarins. Satsuma mandarins were divided into five groups in Japan in the past: the Wase (early season), Zairai (native, indigenous, or old), Owari (a province on Honshu Island), Ikeda and Ikiriki (town or village names) (Hodgson, 1967). They can also be grouped based on their maturity time: Goko Wase (very early), Wase (early), Nakate or Chusei (mid-season) and Bansei (late season) (Saunt, 2000). However, no work has been done so far to study the genetic relationship among the Satsuma mandarins or to fingerprint them based on isozymes or molecular markers. There is a pressing need for the development of reliable methods for identification of Satsuma mandarins and assessment of the genetic diversity among the Satsuma mandarins for future germplasm introduction and maintenance. Study of the genetic relationships among different citrus species was accomplished using different types of markers in the past three decades, markers such as isozymes (Torres et al., 1978; 1982; Hirai et al., 1986; Durham et al., 1992; Jarrell et al., 1992; Fang et al., 1994) Restriction Fragment Length Polymorphism marker (RFLP) (Durham et al., 1992; Jarrell et al., 1992; Cai et al., 1994; Liou et al., 1996; Fang et al., 1997), Random Amplified Polymorphic DNA marker (RAPD) (Cai et al., 1994; Nicolosi et al., 2000), Proc. XXVI IHC IVth Int. Symp. Taxonomy of Cultivated Plants Ed. C.G. Davidson and P. Trehane Acta Hort. 634, ISHS 2004 Publication supported by Can. Int. Dev. Agency (CIDA) 159
Sequence-Characterized Amplified Regions marker (SCARs) (Nicolosi et al., 2000), chloroplast DNA (Nicolosi et al., 2000), Inter-Simple Sequence Repeat (ISSR) (Fang et al., 1997; 1998; Fang and Roose, 1997; Sankar and Moore, 2001; Matsuyama et al., 2001) and Inter-Retrotransposon Amplified Polymorphisms (IRAP) (Bret et al., 2001). The introduction of Amplified Fragment Length Polymorphism (AFLP) as a technique for precision genotyping circumvents all the limitations of previous fingerprinting techniques (Vos et al., 1995). This technique is highly specific, generates a high multiplex ratio and is repeatable unlike RAPD and other markers (Jones et al., 1997). AFLP markers have been used to assess genetic diversity in many tree fruit crops in recent years and it has been shown to be a very powerful marker system for varietal identification. The present study was undertaken to develop AFLP marker system for the identification of Satsuma mandarin cultivars in California. The objectives were 1) to examine the usefulness of AFLPs in differentiating Satsuma mandarin cultivars, and 2) to determine the relationships among the Satsuma mandarin cultivars. MATERIALS AND METHODS Plant Materials The 21 Satsuma mandarin cultivars used in the present study were obtained from the Citrus Clonal Protection Program (CCPP) and the UC Riverside citrus variety collection, UC Riverside (Table 1). Among them, 14 cultivars belong to the Japanese group, four cultivars belong to the Chinese group, and three cultivars are of unknown origin. Also, two citrus species, Citrus yatsushiro (Yatsushiro mikan mandarin) and C. reticulata (Huang yen Man Chieh mandarin) that are closely related to C. unshiu ( Okitsu Wase Satsuma mandarin), and two more distant species, C. aurantium (sour orange) and C. vulgaris were included in the study as controls (Fang et al., 1998). DNA Isolation and AFLP Analysis Total DNA was extracted from young leaves using the CTAB method (Doyle and Doyle, 1987). DNA concentrations were quantified using a Hoefer DyNA Quant200 (Pharmacia Biotech, Piscataway, New Jersey). AFLP analysis was conducted using the GIBRL BRL AFLP System II (Life Technologies, Grand Island, New York) based on a modified protocol following Myburg et al. (2000). The PCR amplification reactions were performed on a MJR Cycle LR TM (MJ Research, Inc., Watertown, Massachusetts). In order to identify the primer combinations revealing clear, reproducible polymorphisms, we screened 64 primer combinations using five Satsuma mandarin cultivars (Frost Nucellar #1 Owari, Okitsu Wase, Dungan, Silverhill and S9). The AFLP products were electrophoresed on 25 cm x 0.25 mm of 8% denaturing polyacrylamide long ranger gel solution (BMA, Rockland, Maine) in 0.8 X TBE buffer using a LI-COR automated sequencer 4000L (LI-COR Inc., Lincoln, Nebrask). Samples were electrophoresed at 1500 V, 50 C for 3.5 hrs. Data Analysis For the genetic similarity analysis, AFLP bands were scored as present (1) or absent (0) to create the binary data set. The data was entered into a binary data matrix as discrete variables. The Dice s coefficient of similarity (Sneath and Sokal, 1973) was calculated for all pair-wise comparisons among the Satsuma mandarin cultivars. A dendrogram was generated by cluster analysis using the unweighted pair group method with the arithmetic average (UPGMA) (NTSYS-pc, version 2.1) (Rohlf, 2000). RESULTS AND DISCUSSION AFLP Profile and Analysis In order to determine the usefulness of AFLP in identifying Satsuma mandarins, we screened 64 EcoR I + 2/Mse I + 3 primer combinations initially using five Satsuma 160
mandarin cultivars (Frost Nucellar #1 Owari, Okitsu Wase, Dungan, Silverhill and S9). We selected six primer combinations for testing on all the samples (E+GG/M+CTA, E+CC/M+CTA, E+GC/M+CTC, E+CA/M+CTC, E+TT/M+CTG, and C+AG/M+CTG). These six primer sets generated a total of 393 scorable products among the 25 genotypes studied, of which 230 bands were polymorphic. AFLP fragment sizes ranging from 50 to 565 bp were scored. Polymorphic fragments were distributed across the entire size range, with the major proportion between 150 and 300 bp. The average percentage of polymorphic bands for the six primer combinations was 58.5%. The highest percentages of polymorphic bands were found in the primer combinations of E+AG/M+CTG (82.6%), followed by E+TT/M+CTG (68.8%), E+CA/M+CTC (58.7%), E+CC/M+CTA (49.1%), E+GG/M+CTC (34.8%), and E+GG/M+CTA (30.0%). The minimum polymorphic bands per primer were found to be 15 for the E+GC/M+CTA and the maximum polymorphic bands were 71 for the primer set E+AG/M+CTG, thus confirming the high multiplex ratio produced by AFLP markers. The data showed that the AFLP markers to be an excellent method for the molecular fingerprinting studies of Satsuma mandarin cultivars due to their ability to pick up large polymorphisms per primer pair. The polymorphisms that observed among the Satsuma mandarins using the AFLP markers were much higher than the ISSR markers (Fang and Roose, 1997). No polymorphism among Frost Owari, Nepolitana, Dobashi Beni, Okitsu Wase, and Kawano Wase Satsuma mandarins was reported using ISSR markers. Identification and Genetic Similarity of Satsuma Mandarins The AFLP data was used to make pair-wise comparisons of the genotypes based on shared and unique amplification products to generate a Dice s coefficient of similarity using the NTSYSpc. The dendrogram of 21 Satsuma cultivars and 4 citrus relatives resulting from a UPGMA cluster analysis based on estimates of Dice s coefficient of similarity obtained from AFLP polymorphism is shown in Fig. 1. Twenty out of 21 Satsuma mandarin cultivars could be classified into a major group when compared with C. yatsushiro, C. reticulata, C. vulgaris, and C. aurantium. These 20 Satsuma mandarins have within-group similarity values of >0.83. Citrus yatsushiro and C. reticulata were more closely related to Satsuma mandarins examined than C. vulgaris and C. aurantium (Fang et al., 1998). Similar phylogenetic relationships among these four species and Satsuma mandarin were also observed in the current study. However, it was surprising to find that the S6 Satsuma mandarin from China was most distant from all other Satsuma mandarins tested. It is possible that the S6 Satsuma may not be a true Satsuma mandarin based on these results. Future field trials will determine the fruit characters of the S6 Satsuma mandarin. The 20 other Satsuma mandarins can be further divided into five subgroups. The first subgroup consists of Frost Nucellar #1 Owari from California and Nepolitana Satsuma from Colombia. The second subgroup consists of the S7 Satsuma from China and Aguzleara Satsuma. The third subgroup includes the Kawano Wase Satsuma. The fourth subgroup consists of the Miho Wase, S2, Aoshima, Iveriya and Dungan Satsuma. The fifth subgroup consists of Dobashi Beni, Okitsu Wase, Miyagawa Wase, S9, Silverhill, Shiroko Listvennyl, Dart North, Dart South, Armstrong and Kuno Wase Satsuma. Overall, we were able to identify 19 individual Satsuma mandarin cultivars out of 21cultivars that we studied. The only pairs that we were unable to differentiate were Dart North and Dart South. The Frost Nucellar #1 Owari was an Owari selection made by H.B. Frost at Rubidoux, CA in 1916. The Nepolitana Owari selection from Colombia is very similar to the Frost Nucellar #1 Owari based on the AFLP polymorphism which suggests these two materials may have similar backgrounds. Six known Owari Satsuma mandarin selections in this study ( Dart North, Dart South, Armstrong, Aoshima, Silverhill, and Dungan ) are distant from the Frost Nucellar #1 Owari selection. Dart North, Dart South and Dungan are Owari selections from California. Armstrong is an Owari selection from Louisiana. The Aoshima is an Owari selection from Japan. 161
Silverhill was a nucellar seedling selection of Owari from a cross made by W.T. Swingle of U.S.D.A. in Florida around 1908 (Hodgson, 1967). Silverhill was introduced back to Japan, then to New Zealand, and finally to California in the 1990 s. Silverhill was once introduced to California from Florida around 1931, but no current planting can be traced to this early introduction. The Dart North, Dart South, and Armstrong are very closely related based on their AFLP polymorphism, together with Kuno Wase from Japan. The Silverhill, Shiroko Listvennyl, S9, Miyagawa Wase, Okitsu Wase and Dobashi Beni are all very similar based on AFLP polymorphism (subgroup 5). All these selections may derive from similar ancestry. The Dobashi Beni and Okitsu Wase are very similar in their polymorphism. The Dobashi Beni was an Owari selection from Japan and the Okitsu Wase was a nucellar seedling of Miyagawa Wase from Japan (Hodgson, 1967). The term Owari Satsuma does not mean a single lineage; rather it indicates these groups of Satsuma mandarin materials came from the Owari region of Japan (currently Aichi prefecture). Many different Owari selections could have been selected initially and distributed throughout Japan and other countries. Multiple introductions of Owari Satsuma from Japan to the United States were made in the early 1900s following the first introduction in 1876 by G.R. Hall (Tanaka, 1932). Over a period of 20 years, more than a half million Owari Satsuma plants were imported to the U.S. from different nurseries in different regions of the Owari region of Japan. The Kuno Wase is most likely an Owari sport selection like many other Wase selections. The parentage of Kuno Wase may derive from material very similar to the parental Owari of Dart North, Dart South, and Armstrong selections. The Dungan, Iveriya, Aoshima, S2, and Miho Wase are similar to each other (subgroup 4). The Miho Wase was a nucellar seedling of Miyagawa Wase, but its AFLP profile is different from the sister seedling of Okitsu Wase. The Kawano Wase was one of the first Wase cultivars found in Japan in the late 1800s (Tanaka, 1932) (subgroup 3). The S7 Satsuma from China and Aguzdera (subgroup 2) both are of unknown parentage. In summary, AFLP markers can be generated efficiently to assess the phylogenetic relationships among Satsuma mandarins. The five subgroups of Satsuma mandarins that we identified in this study can be used as a basis of comparison for other Satsuma mandarins. The AFLP profiles we established in the study can be used for future fingerprinting of Satsuma mandarin cultivars. It also will be helpful for identifying future Satsuma mandarin germplasm introduction and maintenance. ACKNOWLEDGEMENTS We would like to thank Dr. D. Gumpf, Citrus Clonal Protection Program, UC Riverside and Dr. T. Kahn, citrus variety collection, UC Riverside for the assistance in collecting leaf samples for this study. Literature Cited Bret, M.P., Ruiz, C., Pina, J.A. and Asins, M.J. 2001. The diversification of Citrus clementina Hort. ex Tanaka, a vegetatively propagated crop species. Mol. Phyl. Evol. 21:285-293. Cai, Q., Guy, C.L. and Moore, G.A. 1994. Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of coldacclimation-responsive loci. Theor. Appl. Genet. 89:604-614. Doyle, J.J. and Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11-15. Durham, R.E., Liou, P.C., Gmitter, F.G. and Moore, G.A. 1992. Linkage of restriction fragment length polymorphisms and isozymes in Citrus. Theor. Appl. Genet. 84:39-48. Fang, D.Q., Zhang, W.C. and Xiao, S.Y. 1994. Isozymes and classification of Citrus species in China. Acta Bot. Sin. 36:124-138. Fang, D.Q. and Roose, M.L. 1997. Identification of closely related citrus cultivars with inter-simple sequence repeat markers. Theor. Appl. Genet. 93:408-417. 162
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Tables Table 1. Satsuma mandarin cultivar names, Japanese or Chinese type, season (maturity time: early, middle, or late), origin or background of the selection and citrus relatives used as out-group control in the study. Cultivar Name Type Season Origin or Background Kuno Wase Japanese Middle Introduced from South Africa Dart North Japanese Late Owari selection from California Dart South Japanese Late Owari selection from California Dungan Japanese Late Owari selection from California Miho Wase Japanese Early Introduced from New Zealand Miyagawa Wase Japanese Early Introduced from New Zealand Frost nucellar #1 Owari Japanese Late Owari nucellar seedling selection by H.B. Frost in 1916 Nepolitana Japanese Late Introduced from Colombia in 1967 by W. Reuther Kawano Wase Japanese Middle Introduced from Japan before 1928 but its origin is unknown Okitsu Wase Japanese Early Introduced from Japan in 1980 Dobashi Beni Japanese Middle Introduced from Japan in 1979, a sport of Owari from Japan Armstrong Japanese Early Introduced from Louisiana Aoshima Japanese Late Introduced from New Zealand, a mutant of Owari Shiroko Unknown Late? Introduced from New Zealand/Russia Listvennyl Silverhill Japanese Early?- Middle A nucellar seedling of Owari selected by W.T. Swingle in 1908 S2 Chinese Unknown Introduced from Auburn University/China in 1995 S9 Chinese Unknown Introduced from Auburn University/China in 1995 S6 Chinese Unknown Introduced from Auburn University/China in 1995 S7 Chinese Unknown Introduced from Auburn University/China in 1995 Aguzdera Unknown Unknown Introduced from New Zealand Iveriya Unknown Unknown Introduced from New Zealand C. yatsushiro UCR Citrus Variety Collection C. reticulata UCR Citrus Variety Collection C. vulgaris UCR Citrus Variety Collection C. aurantium UCR Citrus Variety Collection 164
Figurese ro 0.71 0.78 0.85 0.92 0.99 Dice coefficient of similarity Kuno Wase Amstrong Dart North Dart South Shiroko Listvennyl Silverhill S9 Miyagawa Wase Okitsu Wase Dobashi Beni Wase Dungan Iveriya Aoshima S2 Miho Wase Kawano Wase S7 Aguzdera Frost Owari Nepolitana C.yatsushiro C.reticulata S6 C.vulgaris C.aurantium Fig. 1. Dendrogram of 21 Satsuma mandarin cultivars, C. yatsushiro, C. reticulata, C. vulgaris, and C. aurantium resulting from a UPGMA cluster analysis based on Dice estimates of similarity obtained from 393 AFLP bands. 165