Identification of Satsuma Mandarin (Citrus unshiu) Cultivars in California Using Amplified Fragment Length Polymorphism (AFLP) Markers

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) 160 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 (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