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An assessment of genetic variability and relationships within Asian pears based on AFLP (amplified fragment length polymorphism) markers
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Scientia Horticulturae 116 (2008) 374–380 www.elsevier.com/locate/scihorti
An assessment of genetic variability and relationships within Asian pears based on AFLP (amplified fragment length polymorphism) markers Lu Bao a, Kunsong Chen a, Dong Zhang a, Xiugen Li b, Yuanwen Teng a,* a
Department of Horticulture, Zhejiang University, The State Agricultural Ministry Laboratory of Horticultural Plant Growth Development & Biotechnology, Hangzhou 310029, China b Zhengzhou Fruit Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, China Received 27 August 2007; received in revised form 14 February 2008; accepted 15 February 2008
Abstract A total of 100 Pyrus L. accessions native mainly to East Asia were subjected to amplified fragment length polymorphism (AFLP) analysis to evaluate genetic variability and relationships among the accessions. Six AFLP primer combinations produced a total of 459 fragments, of which 410 were polymorphic with a polymorphism percentage of 89%. The Dice’s similarity coefficient among pear accessions ranged from 0.671 (P. betulaefolia Bge and P. elaeagrifolia Pall.) to 0.947 (‘Umajirou’ and ‘Immuraaki’). Occidental pears generally had low similarities to Asian pears. The dendrogram generated from all the accessions by unweighted pair-group method of arithmetic analysis (UPGMA) cluster analysis clearly distinguished Occidental pears from accessions of East Asia. P. ussuriensis Maxim., P. betulaefolia and P. communis L. clustered separately into independent groups in accordance with their morphological classification. Japanese pear cultivars formed two groups with some Chinese white pears and Chinese sand pears. Chinese white pears and Chinese sand pears independently formed their own groups and also mingled into mixed groups in the dendrogram. Therefore, Chinese white pears were treated as a cultivated group or an ecotype of P. pyrifolia: P. pyrifolia White Pear Group. The information obtained from this study will be of great help for understanding the origin and evolution of Asian pear cultivars. # 2008 Elsevier B.V. All rights reserved. Keywords: Asian pears; Genetic diversity; AFLP; Pyrus
1. Introduction Pear belongs botanically to the Rosaceae family, subfamily Pomoideae, genus Pyrus L. and is one of the most economically important tree fruit crops in the temperate zones. The Pyrus species, with thousands of cultivars, can be traditionally divided into two native groups according to their geographic distribution: Occidental pears and Oriental pears (Bailey, 1917). Contrasting to the single cultivated species P. communis L. in Occidental pears, at least three species, namely, P. ussuriensis Maxim., P. sinkiangensis Yu and P. pyrifolia Nakai. are involved in the origin of pear cultivars in Oriental pears (Teng and Tanabe, 2004). Based on the geographic distribution and involved species, pear cultivars native to East Asia can be divided into five major groups: Ussurian pears, Chinese white
* Corresponding author. E-mail address: [email protected] (Y. Teng). 0304-4238/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.02.008
pears, Xinjiang pears, Chinese sand pears and Japanese pears. The former four types have been developed in China and the latter one is cultivated in Japan. Ussurian pears have derived from wild P. ussuriensis, naturally distributed in Northeastern China. Xinjiang pear (P. sinkiangensis) is native to Northwestern China and has been proved to be of an interspecific hybridization origin involving Chinese white pears or Chinese sand pears and Occidental pears (Teng et al., 2001). Cultivars of P. sinkangensis vary considerably, combining characteristics of both P. communis and Chinese white pears (Teng and Tanabe, 2004). Chinese sand pear cultivars have undoubtedly originated from P. pyrifolia occurring mainly in Changjiang River valley of China. Chinese white pear cultivars have traditionally been assigned to P. bretschneideri Rehd. by Chinese taxonomists and horticulturists. However, Rubtsov (1944) and Kikuchi (1946) proposed that the origin of Chinese white pears might be involved in the hybridization of P. ussuriensis and P. pyrifolia. Kikuchi (1946) put this group of cultivars under the name of P. ussuriensis Max. var. sinensis
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Kikuchi. As for the origin of Japanese pear cultivars there are also two different points of view. Kikuchi (1948) proposed that Japanese pear cultivars have been domesticated from wild P. pyrifolia distributed in Middle and Southern Japan. On the other hand, some researchers insisted that the progenitor of Japanese pear cultivars might have come from China and Korean peninsula since ancient time (Shimura, 1988; Shirai, 1929). Therefore, the classification of pear cultivars native to East Asia is confusing and the origin of pear cultivars, especially Chinese white pears and Japanese pears is to be solved. Recently, DNA-based molecular markers have been efficiently used for the cultivar discrimination and the analysis of genetic relationships within the genus Pyrus, among which, the most widely used in the previous detailed studies of pears, especially Asian pears, are RFLP (Iketani et al., 1998; Teramoto et al., 1994), RAPD (Teng et al., 2001, 2002), and SSR (Bao et al., 2007; Kimura et al., 2002; Wu¨nsch and Hormaza, 2007). These studies have revealed the genetic difference between Occidental pears and Oriental pears. In addition, a great deal of genetic variability of pear cultivars native to East Asia has been determined, and provided
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interesting insights into the origins of cultivated pears in East Asia. The evidences from RAPD and SSR markers have shown that both Chinese white pears and Japanese pears might have originated directly from P. pyrifolia occurring in China (Bao et al., 2007; Teng et al., 2002; Teng and Tanabe, 2004) and Chinese white pears should be treated as an ecotype of P. pyrifolia (Teng et al., 2002; Teng and Tanabe, 2004). Therefore, it is the first time that Chinese white pears, Chinese sand pears and Japanese pears are designated under a common progenitor. However, further evidence will be needed from different sources, including different DNA markers, to support this hypothesis. The amplified fragment length polymorphism (AFLP) technique (Vos et al., 1995) is based on the PCR amplification of selected restriction fragments of a total genomic DNA digest, which combines the reliability of the RFLP technique with the power and the ease of the PCR technique. Therefore, AFLP is applicable to DNA of any region or complexity without prior sequence information (Vos et al., 1995). Previous studies have used AFLP technique to estimate relationships between a limited selection of pear cultivars, wild forms, and related species (Dofatotowski et al., 2004; Lin et al., 2002; Monte-
Fig. 1. Geographical distribution of the 68 Chinese pear cultivars and types in this study. The map specifies the distribution of each species and cultivar as well as the name of the province. (1) Liaoning, (2) Beijing, (3) Gansu, (4) Qinghai, (5) Hebei, (6) Shandong, (7) Shannxi, (8) Henan, (9) Jiangsu, (10) Hubei, (11) Anhui, (12) Sichuan, (13) Guizhou, (14) Jiangxi, (15) Zhejiang, (16) Fujian, (17) Yunnan, (18) Guangxi, and (19) Northeast China. The geographic distribution of cultivars in China is based on Pu et al. (1989), Pu and Wang (1963); (*) Chinese white pear; (*) Chinese sand pear; (~) P. ussuriensis; (^) wild species originating from East Asia; Classification of species and cultivars is based on Yu (1979).
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Corvo et al., 2000; Pan et al., 2002). Compared to other dominant markers, such as RAPD and ISSR, AFLP showed the largest Marker Index and was the most efficient technique in pear genotyping of P. communis cultivars due to its high capacity to reveal polymorphisms per assay unit (Monte-Corvo et al., 2002). To the best of our knowledge, no detailed research has been conducted to analyze genetic variability and relationships among pear species and cultivars native to East Asia using AFLP markers. In the investigation reported here, large-sized samples of cultivated pears, mainly from China and Japan, were used to assess polymorphism among the accessions using AFLP markers. The objective of this study was to reveal the genetic variability and the relationships among the pear cultivars to understand the origin of cultivated pears in East Asia. 2. Materials and methods 2.1. Plant material A total of 100 Pyrus species and cultivars were used in this study including 27 Chinese sand pear cultivars, 28 Chinese white pear cultivars, 11 Ussurian pear genotypes, 27 Japanese pear cultivars, two types of P. betulaefolia, 2 cultivars of P. communis (‘Bartlett’ and ‘Coscia’) and 3 wild Occidental species (P. amygdaliformis, P. elaeagrifolia and P. longipes).
Figs. 1 and 2 show the geographical distribution of the accessions from East Asia, which were collected from CPGR (China Pear Germplasm Repository, Xingcheng, Liaoning Province, China), TU (Tottori University, Tottori, Japan), ZZFI (Zhengzhou Fruit Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, Henan Province, China), DS (Dangshan County, Anhui Province, China), and HRIYN (Horticultural Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan Province, China). Cultivars of P. communis and wild Occidental species were not included in Figs. 1 and 2. 2.2. DNA extraction and purification protocol Total genomic DNA was extracted from young leaves according to the protocol of Dellaporta et al. (1983) and modified as described by Teng et al. (2002). DNA was purified by following a two-step protocol with a DNA purification kit (SBS Genetech Co., Ltd., China) and re-suspended in TE buffer after washing it with the 70% ethanol. DNA concentration was measured by fluorometry (DU 7500, Beckman) and was adjusted to 100 ng/mL. 2.3. AFLP analysis The AFLP technique was based on Vos et al. (1995) with modifications. The primers used in this study are shown in Table 1. 500 ng of genomic DNA was digested with restriction enzymes EcoRI and MseI at 37 8C for 5 h. Following heat inactivation of the restriction endonucleases, the restricted genomic DNA fragments were ligated to EcoRI and MseI adapters overnight at 16 8C. Digest-ligated DNA fragments were diluted fivefold to be used as templates for the preamplification reaction. This PCR reaction was performed in a total volume of 20 mL containing 1 PCR buffer (with 1.5 mM MgCl2), 2 mM of each dNTP, 10 pmol of each primer (EcoRIA: 50 -GACTGCGTACCAATTC+A-30 and MseI-C:50 -GATGAGTCCTGAGTAA+C-30 ). and 1.0 unit of Taq polymerase (Takara Biotechnology Company, Japan) using the following program: 25 cycles of 94 8C for 30 s, 56 8C for 30 s, and 72 8C for 2 min. The PCR products were diluted 10 times with double distilled water and used as templates for the selective amplification. Six primer combinations were used for selective Table 1 The polymorphic characterization of AFLP primers in Pyrus Primer combination E-AAG/M-CAT E-ACT/M-CTT E-AAC/M-CAG E-AAC/M-CTA E-AAC/M-CAA E-AAG/M-CTC
Fig. 2. Geographical distribution of the 27 Japanese pear cultivars and types collected from TU. The map specifies the distribution of each cultivar of type along with the name of prefecture. (1) Aomori, (2) Niigata, (3) Ishikawa, (4) Hiroshima, (5) Fukuoka, (6) Kanagawa, (7) Nara, (8) Kochi, (9) Kyushu, and (10) unknown. The geographic distribution of cultivars in Japan is based on Jang et al. (1992), Kajiura and Sato (1990), and Kikuchi (1948).
Total Average a b c
Nba
Pbb
67 80 67 99 85 61
56 73 60 87 77 57
459
410
77
68
Number of bands. Polymorphic bands. Percentage of polymorphic bands.
Ppc (%) 83 91 90 88 91 93
89
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amplification (Table 1). Selective amplifications were carried out in a volume of 20 mL containing 5 mL of diluted preamplification PCR product, 10 pmol of each primer, 1.0 unit of Taq polymerase (Takara Biotechnology Company, Japan), 0.2 mM of each dNTP, 1.5 mM MgCl2, and 1 PCR buffer. PCR was performed in an Eppendorf Mastercycler (Gradient, no. 5331-41264, Germany) with the following touch-down program: 32 cycles of 94 8C for 30 s, annealing (see below) for 30 s, and 72 8C for 2 min with a final extension at 60 8C for 30 min. Annealing was initially at 65 8C and decreased by 0.7 8C per cycle for eight cycles, remaining at 57 8C for the remaining 23 cycles. Twenty microliters of PCR product was mixed with 3 mL formamide loading buffer (98% formamide, 10 mM EDTA, 0.005% bromophenol blue, 0.005% xylene cyanol, pH 8.0) and was subsequently heat-denatured at 95 8C for 5 min. Five microliters of each mixture and a molecular-weight marker (DingGuo Biotechnology Company, China) was loaded onto a 6% (0.4-mm thick) polyacrylamide gel (7 M urea) in 1 TBE buffer (90 mM Tris–borate, 2 mM EDTA, and pH 8.3). The gel was run at a constant 75 W, 45 8C for 3 h in a sequencing gel electrophoresis apparatus (Bio-Rad 3000, US). The primers were not radioactively labeled; therefore, AFLP fragments were visualized using silver staining (Brant, 1991). Only bands which were consistent over independent amplifications were considered in this study. 2.4. Data analysis For the genetic relationship analysis, each gel was analyzed by manually scoring the presence (1) or absence (0) of bands in individual lanes. Only reproducible bands were used to calculate Dice’s similarity coefficients (Nei and Li, 1979). A dendrogram was constructed based on the similarity matrix by unweighted pair-group method of arithmetic analysis (UPGMA), using the NTSYS-pc program (version 2.02j) (Rolf, 1998). 3. Results 3.1. AFLP polymorphism Amplification of genomic DNA of the 100 accessions, using six primer combinations for AFLP analysis produced a total of 459 scorable fragments ranging in size from 70 to 1000 bp. Of these, 410 fragments were polymorphic. The number of bands and the degree of polymorphism, revealed by each primer combination, is given in Table 1. EcoRI-AAG/MseI-CTC showed the highest percentage of polymorphic bands and EcoRI-AAC/MseI-CTA lowest. 3.2. Genetic relationships among Pyrus species and cultivars The similarity coefficient, estimated by Dice’s coefficient (Nei and Li, 1979), ranged from 0.671 for P. betulaefolia and P. elaeagrifolia to 0.947 for ‘Umajirou’ and ‘Imamuraaki’.
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Species and cultivars native to East Asia had low similarities to the cultivars of P. communis and Occidental species, while the genotypes native to East Asia generally had high degree of genetic similarity to each other. Genetic relationships among the 100 accessions, based on their genetic similarities, were revealed in a dendrogram resulting from the UPGMA cluster analysis (Fig. 3). The dendrogram clearly distinguished Occidental pears including cultivars of P. communis from accessions native to East Asia at the 0.77 level of similarity index (Fig. 3). The pear accessions from East Asia were further divided into 13 groups (Groups 1– 13) at the 0.88 level of similarity index, and the Occidental pears could be divided into two distinct groups (Groups 14 and 15). A majority of the Japanese pear cultivars fell into Group 1 along with some Chinese white pear cultivars and a few Chinese sand pear cultivars, and two remaining Japanese cultivars ‘Hakataao’ and ‘Kopeitao’ were clustered into Group 6 with a Chinese sand pear cultivar and a Chinese white pear cultivar. Within Group 1, six subgroups (I–VI) could be identified (Fig. 3). Japanese pear cultivars were included in subgroups II and V. Subgroup II consisted of all Japanese pears, but in subgroup V a Chinese sand pear cultivar ‘Puguali’ was also included. The other subgroups were composed of Chinese white pear cultivars and/or Chinese sand pear cultivars. All the cultivars of P. ussuriensis clustered independently into two groups (Groups 2 and 5). In Group 2, ‘Beijingbaili’, a famous P. ussuriensis cultivar native to Beijing distantly clustered with other cultivars native to Liaoning Province. ‘Yeli’, a wild type of P. ussuriensis seemed to have a large genetic distance to the cultivars. Like Group 2, Group 5 was also composed of cultivars from Liaoning Province and the cultivar ‘Yaguangli’ originating from Beijing. Chinese white pears generally mingled with Chinese sand pears to form groups or were clustered individually (Fig. 3). They were concentrated in Groups 4, 7, and 10 and also scattered in Groups 1, 3, 6, 8, 9, and 12 with Chinese sand pears and/or Japanese pears. Chinese sand pears were mainly included in Groups 8, 9, and 11 (Fig. 3). Two genotypes of P. betulaefolia Bge. from different places clustered together into Group 13 and branched distantly from the majority of Chinese white pears, Chinese sand pears, Japanese pears and Ussurian pears. Two P. communis cultivars ‘Bartlett’ from England and ‘Coscia’ from Russia clustered distantly into Group 14. P. amygdaliformis and P. elaeagrifolia, both native to south Europe, clustered first and then further grouped with P. longipes originating from North Africa. 4. Discussion AFLP was demonstrated to be the most efficient technique in pear genotyping of P. conmmunis cultivars (Monte-Corvo et al., 2002). To our knowledge, this is the first detailed study to use AFLP for examining the genetic variability and relationships of Asian pear species and cultivars. Some accessions used in this study had been evaluated using RAPD markers (Teng et al.,
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Fig. 3. Dendrogram of 100 pear species and cultivars resulting from UPGMA analysis based on Dice’s similarity coefficient. (*) Chinese white pear; (*) Chinese sand pear; (D) Japanese pear; (~) P. ussuriensis; (^) wild species originating from East Asia; ( ) Cultivars of P. communis; ( ) Occidental pear species.
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2001, 2002) and SSR markers (Bao et al., 2007), but have never been examined by AFLP markers. The present study demonstrated that the percentage of polymorphic bands was as high as 89%, which is in accordance with the similar levels of molecular polymorphisms (87%) previously reported by Monte-Corvo et al. (2000). Nei and Li’s (1979) similarity coefficient values among 100 pear cultivars from the present AFLP data are higher than those from the RAPD (Teng et al., 2002) and SSR data (Bao et al., 2007). Similar results were also reported in a strawberry study using both RAPD and AFLP markers (Degani et al., 2001) and an apple study using RAPD, AFLP and SSR markers (Goula˜o and Oliveira, 2001). These researches indicate a higher percentage of bands shared in AFLP markers than those shared in RAPD or SSR markers. Considering both He and similarity coefficient, SSR are more appropriate for pear identification purposes, since they provide a higher level of polymorphism among the samples, AFLP, however, is more suitable for revealing genetic relationships of pears because of a higher sharing of bands in AFLP markers. The genetic relationships revealed by the dendrogram (Fig. 3) are in good agreement with the results of Oliveira et al. (1999), Monte-Corvo et al. (2000) and Teng et al. (2001, 2002) who also divided Pyrus into the Occidental species and the Oriental species using AFLP or RAPD markers. These results support the traditional view that genus Pyrus consists of two geographic species groups: Occidental pears and Oriental pears (Bailey, 1917). Japanese pears have been considered to be the same germplasm as Chinese sand pears and grouped into P. pyrifolia. The morphological characteristics of Japanese pear cultivars are similar to those of Chinese sand pears (Teng and Tanabe, 2004). In this study, Japanese pears clustered with some Chinese sand pears and Chinese white pears (Fig. 3), especially those originating from Zhejiang Province and its bordering provinces (Fig. 1); this is the same result as that using SSR markers (Bao et al., 2007). Our previous study with RAPD markers also found that those from Zhejiang Province were close to some Japanese pear cultivars. All the accessions originating from Kochi Prefecture of Japan (Fig. 2) did not cluster together, but scattered into two subgroups of Group 1 (Fig. 3), indicating a large genetic diversity, which supports our previous view that the germplasm native to Kochi Prefecture is related to other populations of Japanese pears (Teng et al., 2002). Teng et al. (2002) proved that some Japanese pear cultivars from Kochi Prefecture had similarities to some Chinese sand pear cultivars, especially from Zhejiang and Fujian Provinces. They speculated those might have been introduced from ancient China, since a flourishing sea route existed for trade and cultural exchanges between Kochi Prefecture of Japan and Zhejiang Province of China (Yoneyama, 2001). Our recent series of studies with different DNA markers (Bao et al., 2007; Shen et al., 2006; Teng et al., 2001, 2002), including AFLP in this study, infer that Japanese pears might have developed from progenitor genotypes coming from ancient China. Ussurian pears formed two groups (Fig. 3), which are different from our previous SSR work, where all Ussurian pears
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clustered together. However, Ussurian pear cultivars in both studies were not mingled with other types of cultivars, such as Chinese white pears. The RAPD markers specific to P. ussuriensis were generally not found in Chinese white pears (Teng et al., 2002). Morphologically and physiologically, Ussurian pear cultivars differ very much from other cultivated large-sized pear types native to East Asia. The fruit of Ussurian pears are usually small globose and oblate with a persistent calyx, and become soft and edible after a ripening period. This is clearly different from the divergent shapes of fruit with a deciduous calyx and the crisp flesh texture of Chinese white pears, Chinese sand pears, and Japanese pears (Yu, 1979). Both the marker and morphological data would infer that Chinese white pear cultivars could not be a variety of P. ussuriensis and do not support Kikuchi’s nomenclature of Chinese white pears: P. ussuriensis var. sinensis. Chinese white pears and sand pears are the most cultivated pears in North China and South China, respectively. Chinese white pears and Chinese sand pears could independently form their own groups, but mingled mostly into mixed groups in the dendrogram (Fig. 3). This kind of grouping is similar to our previous results with RAPD makers (Teng et al., 2002) and SSR markers (Bao et al., 2007). Both kinds of pears also have similar peroxidase isozymic pattern (Lin and Shen, 1983) and pollen ultrastructure (Zou et al., 1986). These results could very well represent the close genetic relationship between Chinese white pears and Chinese sand pears. Chinese white pear cultivars are morphologically much like Chinese sand pears, making taxonomists unable to classify the cultivars of Chinese pears. In this study some Chinese white pear cultivars from Anhui Province, the northern border of the natural distribution of Chinese sand pears, independently clustered together into Group 4, which reflect that pear cultivars derived from the same geographic location generally had close relationships. Because they are physiologically closer to Chinese sand pears, some authors also classify them as Chinese sand pears (Pu et al., 1989). P. betulaefolia Bge. is distributed in North China (Yu, 1979) and has been extensively used for pear rootstocks in East Asia. Two clones from different places clustered together and just located between Occidental pears and other Oriental pears in dendrogram, which is consistent with the results obtained from RAPD markers (Teng et al., 2002). The position of P. betulaefolia in dendrogram resulting from both RAPD and AFLP data may indicate that it would be a transitional species between the Occidental pears and Oriental pears, as having been proposed by Challice and Westwood (1973). In summary, a high level of genetic variability among the Pyrus species and cultivars mainly native to East Asia was revealed by AFLP markers. Our results confirmed the close relationship between Chinese white pears and Chinese sand pears. Therefore, we treated the Chinese white pears as a cultivated group or an ecotype of Chinese sand pears: P. pyrifolia White Pear Group. The data in this study also suggest that the progenitor of Japanese pear might have Chinese origin. The information obtained from this study will be useful for understanding the origin and evolution of Asian pear cultivars.
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Until now, studies on the origin and development of the cultivated pears in Asia have been rather limited. In the future, analyses of wild populations related to the development of cultivated pear will be needed to understand the origin of Asian pear cultivars. Acknowledgements This research was funded by National Natural Science Foundation of China, grant No. 30370988 and by Zhejiang Provincial Natural Science Foundation of China, grant No. R307605. We greatly acknowledge Prof. Rajagopalan Sridhar of Howard University, USA, for his critical reading of this manuscript. References Bailey, L.H., 1917. Pyrus. Standard Cyclopedia of Horticulture, vol. V. Macmillan, New York, pp. 2865–2878. Bao, L., Chen, K.S., Zhang, D., Cao, Y.F., Yamamoto, T., Teng, Y.W., 2007. Genetic diversity and similarity of pear cultivars native to East Asia revealed by SSR (simple sequence repeat) markers. Genet. Resour. Crop Evol. 54, 959–971. Brant, J., 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 195, 80–83. Challice, J.S., Westwood, M.N., 1973. Numerical taxonomic studies of the genus Pyrus using both chemical and botanical characters. Bot. J. Linn. Soc. 67, 121–148. Degani, C., Rowland, L.J., Saunders, J.A., Hokanson, S.C., Ogden, E.L., GolanGoldhirsh, A., Galletta, G.J., 2001. A comparison of genetic relationship measures in strawberry (Fragaria ananassa Duch.) based on AFLPs, RAPDs, and pedigree data. Euphytica 117, 1–12. Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1, 19–21. Dofatotowski, J., Nowosiefski, J., Podyma, W., Szymansku, M., Zych, M., 2004. Molecular studies on the variability of Polish semi-wild pears (Pyrus) using AFLP. J. Fruit Ornamental Plant Res. 12, 331–337. Goula˜o, L., Oliveira, C.M., 2001. Molecular characterisation of cultivars of apple (Malus domestica Borkh.) using microsatellite (SSR and ISSR) markers. Euphytica 122, 81–89. Iketani, H., Manabe, T., Matsuta, N., Akihama, T., Hayashi, T., 1998. Incongruence between RFLPs of chloroplast DNA and morphological classification in East Asia pear (Pyrus spp.). Genet. Resour. Crop Evol. 45, 533–539. Jang, J.T., Tanabe, K., Tamura, F., Banno, K., 1992. Identification of Pyrus species by leaf peroxidase isozyme pheotypes. J. Jpn. Soc. Hort. Sci. 61, 273–286 (in Japanese with English summary). Kajiura, I., Sato, Y., 1990. Recent progress in Japanese pear (Pyrus pyrifolia Nakai) breeding and descriptions of cultivars based on literature review (in Japanese with English summary). Bull. Fruit Tree Res. 1–329 (Sta. Extra no. 1). Kikuchi, A., 1946. Assessment of Chinese pear species and cultivars. Collected Records Horticulture Research, vol. 3. Faculty of Agriculture, Kyoto, Japan, pp. 1–11 (in Japanese). Kikuchi, A., 1948. Horticulture of Fruit Trees (in Japanese), vol. 1. Yokendo, Tokyo.
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Scientia Horticulturae 116 (2008) 374–380 www.elsevier.com/locate/scihorti
An assessment of genetic variability and relationships within Asian pears based on AFLP (amplified fragment length polymorphism) markers Lu Bao a, Kunsong Chen a, Dong Zhang a, Xiugen Li b, Yuanwen Teng a,* a
Department of Horticulture, Zhejiang University, The State Agricultural Ministry Laboratory of Horticultural Plant Growth Development & Biotechnology, Hangzhou 310029, China b Zhengzhou Fruit Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, China Received 27 August 2007; received in revised form 14 February 2008; accepted 15 February 2008
Abstract A total of 100 Pyrus L. accessions native mainly to East Asia were subjected to amplified fragment length polymorphism (AFLP) analysis to evaluate genetic variability and relationships among the accessions. Six AFLP primer combinations produced a total of 459 fragments, of which 410 were polymorphic with a polymorphism percentage of 89%. The Dice’s similarity coefficient among pear accessions ranged from 0.671 (P. betulaefolia Bge and P. elaeagrifolia Pall.) to 0.947 (‘Umajirou’ and ‘Immuraaki’). Occidental pears generally had low similarities to Asian pears. The dendrogram generated from all the accessions by unweighted pair-group method of arithmetic analysis (UPGMA) cluster analysis clearly distinguished Occidental pears from accessions of East Asia. P. ussuriensis Maxim., P. betulaefolia and P. communis L. clustered separately into independent groups in accordance with their morphological classification. Japanese pear cultivars formed two groups with some Chinese white pears and Chinese sand pears. Chinese white pears and Chinese sand pears independently formed their own groups and also mingled into mixed groups in the dendrogram. Therefore, Chinese white pears were treated as a cultivated group or an ecotype of P. pyrifolia: P. pyrifolia White Pear Group. The information obtained from this study will be of great help for understanding the origin and evolution of Asian pear cultivars. # 2008 Elsevier B.V. All rights reserved. Keywords: Asian pears; Genetic diversity; AFLP; Pyrus
1. Introduction Pear belongs botanically to the Rosaceae family, subfamily Pomoideae, genus Pyrus L. and is one of the most economically important tree fruit crops in the temperate zones. The Pyrus species, with thousands of cultivars, can be traditionally divided into two native groups according to their geographic distribution: Occidental pears and Oriental pears (Bailey, 1917). Contrasting to the single cultivated species P. communis L. in Occidental pears, at least three species, namely, P. ussuriensis Maxim., P. sinkiangensis Yu and P. pyrifolia Nakai. are involved in the origin of pear cultivars in Oriental pears (Teng and Tanabe, 2004). Based on the geographic distribution and involved species, pear cultivars native to East Asia can be divided into five major groups: Ussurian pears, Chinese white
* Corresponding author. E-mail address: [email protected] (Y. Teng). 0304-4238/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.02.008
pears, Xinjiang pears, Chinese sand pears and Japanese pears. The former four types have been developed in China and the latter one is cultivated in Japan. Ussurian pears have derived from wild P. ussuriensis, naturally distributed in Northeastern China. Xinjiang pear (P. sinkiangensis) is native to Northwestern China and has been proved to be of an interspecific hybridization origin involving Chinese white pears or Chinese sand pears and Occidental pears (Teng et al., 2001). Cultivars of P. sinkangensis vary considerably, combining characteristics of both P. communis and Chinese white pears (Teng and Tanabe, 2004). Chinese sand pear cultivars have undoubtedly originated from P. pyrifolia occurring mainly in Changjiang River valley of China. Chinese white pear cultivars have traditionally been assigned to P. bretschneideri Rehd. by Chinese taxonomists and horticulturists. However, Rubtsov (1944) and Kikuchi (1946) proposed that the origin of Chinese white pears might be involved in the hybridization of P. ussuriensis and P. pyrifolia. Kikuchi (1946) put this group of cultivars under the name of P. ussuriensis Max. var. sinensis
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Kikuchi. As for the origin of Japanese pear cultivars there are also two different points of view. Kikuchi (1948) proposed that Japanese pear cultivars have been domesticated from wild P. pyrifolia distributed in Middle and Southern Japan. On the other hand, some researchers insisted that the progenitor of Japanese pear cultivars might have come from China and Korean peninsula since ancient time (Shimura, 1988; Shirai, 1929). Therefore, the classification of pear cultivars native to East Asia is confusing and the origin of pear cultivars, especially Chinese white pears and Japanese pears is to be solved. Recently, DNA-based molecular markers have been efficiently used for the cultivar discrimination and the analysis of genetic relationships within the genus Pyrus, among which, the most widely used in the previous detailed studies of pears, especially Asian pears, are RFLP (Iketani et al., 1998; Teramoto et al., 1994), RAPD (Teng et al., 2001, 2002), and SSR (Bao et al., 2007; Kimura et al., 2002; Wu¨nsch and Hormaza, 2007). These studies have revealed the genetic difference between Occidental pears and Oriental pears. In addition, a great deal of genetic variability of pear cultivars native to East Asia has been determined, and provided
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interesting insights into the origins of cultivated pears in East Asia. The evidences from RAPD and SSR markers have shown that both Chinese white pears and Japanese pears might have originated directly from P. pyrifolia occurring in China (Bao et al., 2007; Teng et al., 2002; Teng and Tanabe, 2004) and Chinese white pears should be treated as an ecotype of P. pyrifolia (Teng et al., 2002; Teng and Tanabe, 2004). Therefore, it is the first time that Chinese white pears, Chinese sand pears and Japanese pears are designated under a common progenitor. However, further evidence will be needed from different sources, including different DNA markers, to support this hypothesis. The amplified fragment length polymorphism (AFLP) technique (Vos et al., 1995) is based on the PCR amplification of selected restriction fragments of a total genomic DNA digest, which combines the reliability of the RFLP technique with the power and the ease of the PCR technique. Therefore, AFLP is applicable to DNA of any region or complexity without prior sequence information (Vos et al., 1995). Previous studies have used AFLP technique to estimate relationships between a limited selection of pear cultivars, wild forms, and related species (Dofatotowski et al., 2004; Lin et al., 2002; Monte-
Fig. 1. Geographical distribution of the 68 Chinese pear cultivars and types in this study. The map specifies the distribution of each species and cultivar as well as the name of the province. (1) Liaoning, (2) Beijing, (3) Gansu, (4) Qinghai, (5) Hebei, (6) Shandong, (7) Shannxi, (8) Henan, (9) Jiangsu, (10) Hubei, (11) Anhui, (12) Sichuan, (13) Guizhou, (14) Jiangxi, (15) Zhejiang, (16) Fujian, (17) Yunnan, (18) Guangxi, and (19) Northeast China. The geographic distribution of cultivars in China is based on Pu et al. (1989), Pu and Wang (1963); (*) Chinese white pear; (*) Chinese sand pear; (~) P. ussuriensis; (^) wild species originating from East Asia; Classification of species and cultivars is based on Yu (1979).
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Corvo et al., 2000; Pan et al., 2002). Compared to other dominant markers, such as RAPD and ISSR, AFLP showed the largest Marker Index and was the most efficient technique in pear genotyping of P. communis cultivars due to its high capacity to reveal polymorphisms per assay unit (Monte-Corvo et al., 2002). To the best of our knowledge, no detailed research has been conducted to analyze genetic variability and relationships among pear species and cultivars native to East Asia using AFLP markers. In the investigation reported here, large-sized samples of cultivated pears, mainly from China and Japan, were used to assess polymorphism among the accessions using AFLP markers. The objective of this study was to reveal the genetic variability and the relationships among the pear cultivars to understand the origin of cultivated pears in East Asia. 2. Materials and methods 2.1. Plant material A total of 100 Pyrus species and cultivars were used in this study including 27 Chinese sand pear cultivars, 28 Chinese white pear cultivars, 11 Ussurian pear genotypes, 27 Japanese pear cultivars, two types of P. betulaefolia, 2 cultivars of P. communis (‘Bartlett’ and ‘Coscia’) and 3 wild Occidental species (P. amygdaliformis, P. elaeagrifolia and P. longipes).
Figs. 1 and 2 show the geographical distribution of the accessions from East Asia, which were collected from CPGR (China Pear Germplasm Repository, Xingcheng, Liaoning Province, China), TU (Tottori University, Tottori, Japan), ZZFI (Zhengzhou Fruit Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, Henan Province, China), DS (Dangshan County, Anhui Province, China), and HRIYN (Horticultural Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan Province, China). Cultivars of P. communis and wild Occidental species were not included in Figs. 1 and 2. 2.2. DNA extraction and purification protocol Total genomic DNA was extracted from young leaves according to the protocol of Dellaporta et al. (1983) and modified as described by Teng et al. (2002). DNA was purified by following a two-step protocol with a DNA purification kit (SBS Genetech Co., Ltd., China) and re-suspended in TE buffer after washing it with the 70% ethanol. DNA concentration was measured by fluorometry (DU 7500, Beckman) and was adjusted to 100 ng/mL. 2.3. AFLP analysis The AFLP technique was based on Vos et al. (1995) with modifications. The primers used in this study are shown in Table 1. 500 ng of genomic DNA was digested with restriction enzymes EcoRI and MseI at 37 8C for 5 h. Following heat inactivation of the restriction endonucleases, the restricted genomic DNA fragments were ligated to EcoRI and MseI adapters overnight at 16 8C. Digest-ligated DNA fragments were diluted fivefold to be used as templates for the preamplification reaction. This PCR reaction was performed in a total volume of 20 mL containing 1 PCR buffer (with 1.5 mM MgCl2), 2 mM of each dNTP, 10 pmol of each primer (EcoRIA: 50 -GACTGCGTACCAATTC+A-30 and MseI-C:50 -GATGAGTCCTGAGTAA+C-30 ). and 1.0 unit of Taq polymerase (Takara Biotechnology Company, Japan) using the following program: 25 cycles of 94 8C for 30 s, 56 8C for 30 s, and 72 8C for 2 min. The PCR products were diluted 10 times with double distilled water and used as templates for the selective amplification. Six primer combinations were used for selective Table 1 The polymorphic characterization of AFLP primers in Pyrus Primer combination E-AAG/M-CAT E-ACT/M-CTT E-AAC/M-CAG E-AAC/M-CTA E-AAC/M-CAA E-AAG/M-CTC
Fig. 2. Geographical distribution of the 27 Japanese pear cultivars and types collected from TU. The map specifies the distribution of each cultivar of type along with the name of prefecture. (1) Aomori, (2) Niigata, (3) Ishikawa, (4) Hiroshima, (5) Fukuoka, (6) Kanagawa, (7) Nara, (8) Kochi, (9) Kyushu, and (10) unknown. The geographic distribution of cultivars in Japan is based on Jang et al. (1992), Kajiura and Sato (1990), and Kikuchi (1948).
Total Average a b c
Nba
Pbb
67 80 67 99 85 61
56 73 60 87 77 57
459
410
77
68
Number of bands. Polymorphic bands. Percentage of polymorphic bands.
Ppc (%) 83 91 90 88 91 93
89
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amplification (Table 1). Selective amplifications were carried out in a volume of 20 mL containing 5 mL of diluted preamplification PCR product, 10 pmol of each primer, 1.0 unit of Taq polymerase (Takara Biotechnology Company, Japan), 0.2 mM of each dNTP, 1.5 mM MgCl2, and 1 PCR buffer. PCR was performed in an Eppendorf Mastercycler (Gradient, no. 5331-41264, Germany) with the following touch-down program: 32 cycles of 94 8C for 30 s, annealing (see below) for 30 s, and 72 8C for 2 min with a final extension at 60 8C for 30 min. Annealing was initially at 65 8C and decreased by 0.7 8C per cycle for eight cycles, remaining at 57 8C for the remaining 23 cycles. Twenty microliters of PCR product was mixed with 3 mL formamide loading buffer (98% formamide, 10 mM EDTA, 0.005% bromophenol blue, 0.005% xylene cyanol, pH 8.0) and was subsequently heat-denatured at 95 8C for 5 min. Five microliters of each mixture and a molecular-weight marker (DingGuo Biotechnology Company, China) was loaded onto a 6% (0.4-mm thick) polyacrylamide gel (7 M urea) in 1 TBE buffer (90 mM Tris–borate, 2 mM EDTA, and pH 8.3). The gel was run at a constant 75 W, 45 8C for 3 h in a sequencing gel electrophoresis apparatus (Bio-Rad 3000, US). The primers were not radioactively labeled; therefore, AFLP fragments were visualized using silver staining (Brant, 1991). Only bands which were consistent over independent amplifications were considered in this study. 2.4. Data analysis For the genetic relationship analysis, each gel was analyzed by manually scoring the presence (1) or absence (0) of bands in individual lanes. Only reproducible bands were used to calculate Dice’s similarity coefficients (Nei and Li, 1979). A dendrogram was constructed based on the similarity matrix by unweighted pair-group method of arithmetic analysis (UPGMA), using the NTSYS-pc program (version 2.02j) (Rolf, 1998). 3. Results 3.1. AFLP polymorphism Amplification of genomic DNA of the 100 accessions, using six primer combinations for AFLP analysis produced a total of 459 scorable fragments ranging in size from 70 to 1000 bp. Of these, 410 fragments were polymorphic. The number of bands and the degree of polymorphism, revealed by each primer combination, is given in Table 1. EcoRI-AAG/MseI-CTC showed the highest percentage of polymorphic bands and EcoRI-AAC/MseI-CTA lowest. 3.2. Genetic relationships among Pyrus species and cultivars The similarity coefficient, estimated by Dice’s coefficient (Nei and Li, 1979), ranged from 0.671 for P. betulaefolia and P. elaeagrifolia to 0.947 for ‘Umajirou’ and ‘Imamuraaki’.
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Species and cultivars native to East Asia had low similarities to the cultivars of P. communis and Occidental species, while the genotypes native to East Asia generally had high degree of genetic similarity to each other. Genetic relationships among the 100 accessions, based on their genetic similarities, were revealed in a dendrogram resulting from the UPGMA cluster analysis (Fig. 3). The dendrogram clearly distinguished Occidental pears including cultivars of P. communis from accessions native to East Asia at the 0.77 level of similarity index (Fig. 3). The pear accessions from East Asia were further divided into 13 groups (Groups 1– 13) at the 0.88 level of similarity index, and the Occidental pears could be divided into two distinct groups (Groups 14 and 15). A majority of the Japanese pear cultivars fell into Group 1 along with some Chinese white pear cultivars and a few Chinese sand pear cultivars, and two remaining Japanese cultivars ‘Hakataao’ and ‘Kopeitao’ were clustered into Group 6 with a Chinese sand pear cultivar and a Chinese white pear cultivar. Within Group 1, six subgroups (I–VI) could be identified (Fig. 3). Japanese pear cultivars were included in subgroups II and V. Subgroup II consisted of all Japanese pears, but in subgroup V a Chinese sand pear cultivar ‘Puguali’ was also included. The other subgroups were composed of Chinese white pear cultivars and/or Chinese sand pear cultivars. All the cultivars of P. ussuriensis clustered independently into two groups (Groups 2 and 5). In Group 2, ‘Beijingbaili’, a famous P. ussuriensis cultivar native to Beijing distantly clustered with other cultivars native to Liaoning Province. ‘Yeli’, a wild type of P. ussuriensis seemed to have a large genetic distance to the cultivars. Like Group 2, Group 5 was also composed of cultivars from Liaoning Province and the cultivar ‘Yaguangli’ originating from Beijing. Chinese white pears generally mingled with Chinese sand pears to form groups or were clustered individually (Fig. 3). They were concentrated in Groups 4, 7, and 10 and also scattered in Groups 1, 3, 6, 8, 9, and 12 with Chinese sand pears and/or Japanese pears. Chinese sand pears were mainly included in Groups 8, 9, and 11 (Fig. 3). Two genotypes of P. betulaefolia Bge. from different places clustered together into Group 13 and branched distantly from the majority of Chinese white pears, Chinese sand pears, Japanese pears and Ussurian pears. Two P. communis cultivars ‘Bartlett’ from England and ‘Coscia’ from Russia clustered distantly into Group 14. P. amygdaliformis and P. elaeagrifolia, both native to south Europe, clustered first and then further grouped with P. longipes originating from North Africa. 4. Discussion AFLP was demonstrated to be the most efficient technique in pear genotyping of P. conmmunis cultivars (Monte-Corvo et al., 2002). To our knowledge, this is the first detailed study to use AFLP for examining the genetic variability and relationships of Asian pear species and cultivars. Some accessions used in this study had been evaluated using RAPD markers (Teng et al.,
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Fig. 3. Dendrogram of 100 pear species and cultivars resulting from UPGMA analysis based on Dice’s similarity coefficient. (*) Chinese white pear; (*) Chinese sand pear; (D) Japanese pear; (~) P. ussuriensis; (^) wild species originating from East Asia; ( ) Cultivars of P. communis; ( ) Occidental pear species.
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2001, 2002) and SSR markers (Bao et al., 2007), but have never been examined by AFLP markers. The present study demonstrated that the percentage of polymorphic bands was as high as 89%, which is in accordance with the similar levels of molecular polymorphisms (87%) previously reported by Monte-Corvo et al. (2000). Nei and Li’s (1979) similarity coefficient values among 100 pear cultivars from the present AFLP data are higher than those from the RAPD (Teng et al., 2002) and SSR data (Bao et al., 2007). Similar results were also reported in a strawberry study using both RAPD and AFLP markers (Degani et al., 2001) and an apple study using RAPD, AFLP and SSR markers (Goula˜o and Oliveira, 2001). These researches indicate a higher percentage of bands shared in AFLP markers than those shared in RAPD or SSR markers. Considering both He and similarity coefficient, SSR are more appropriate for pear identification purposes, since they provide a higher level of polymorphism among the samples, AFLP, however, is more suitable for revealing genetic relationships of pears because of a higher sharing of bands in AFLP markers. The genetic relationships revealed by the dendrogram (Fig. 3) are in good agreement with the results of Oliveira et al. (1999), Monte-Corvo et al. (2000) and Teng et al. (2001, 2002) who also divided Pyrus into the Occidental species and the Oriental species using AFLP or RAPD markers. These results support the traditional view that genus Pyrus consists of two geographic species groups: Occidental pears and Oriental pears (Bailey, 1917). Japanese pears have been considered to be the same germplasm as Chinese sand pears and grouped into P. pyrifolia. The morphological characteristics of Japanese pear cultivars are similar to those of Chinese sand pears (Teng and Tanabe, 2004). In this study, Japanese pears clustered with some Chinese sand pears and Chinese white pears (Fig. 3), especially those originating from Zhejiang Province and its bordering provinces (Fig. 1); this is the same result as that using SSR markers (Bao et al., 2007). Our previous study with RAPD markers also found that those from Zhejiang Province were close to some Japanese pear cultivars. All the accessions originating from Kochi Prefecture of Japan (Fig. 2) did not cluster together, but scattered into two subgroups of Group 1 (Fig. 3), indicating a large genetic diversity, which supports our previous view that the germplasm native to Kochi Prefecture is related to other populations of Japanese pears (Teng et al., 2002). Teng et al. (2002) proved that some Japanese pear cultivars from Kochi Prefecture had similarities to some Chinese sand pear cultivars, especially from Zhejiang and Fujian Provinces. They speculated those might have been introduced from ancient China, since a flourishing sea route existed for trade and cultural exchanges between Kochi Prefecture of Japan and Zhejiang Province of China (Yoneyama, 2001). Our recent series of studies with different DNA markers (Bao et al., 2007; Shen et al., 2006; Teng et al., 2001, 2002), including AFLP in this study, infer that Japanese pears might have developed from progenitor genotypes coming from ancient China. Ussurian pears formed two groups (Fig. 3), which are different from our previous SSR work, where all Ussurian pears
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clustered together. However, Ussurian pear cultivars in both studies were not mingled with other types of cultivars, such as Chinese white pears. The RAPD markers specific to P. ussuriensis were generally not found in Chinese white pears (Teng et al., 2002). Morphologically and physiologically, Ussurian pear cultivars differ very much from other cultivated large-sized pear types native to East Asia. The fruit of Ussurian pears are usually small globose and oblate with a persistent calyx, and become soft and edible after a ripening period. This is clearly different from the divergent shapes of fruit with a deciduous calyx and the crisp flesh texture of Chinese white pears, Chinese sand pears, and Japanese pears (Yu, 1979). Both the marker and morphological data would infer that Chinese white pear cultivars could not be a variety of P. ussuriensis and do not support Kikuchi’s nomenclature of Chinese white pears: P. ussuriensis var. sinensis. Chinese white pears and sand pears are the most cultivated pears in North China and South China, respectively. Chinese white pears and Chinese sand pears could independently form their own groups, but mingled mostly into mixed groups in the dendrogram (Fig. 3). This kind of grouping is similar to our previous results with RAPD makers (Teng et al., 2002) and SSR markers (Bao et al., 2007). Both kinds of pears also have similar peroxidase isozymic pattern (Lin and Shen, 1983) and pollen ultrastructure (Zou et al., 1986). These results could very well represent the close genetic relationship between Chinese white pears and Chinese sand pears. Chinese white pear cultivars are morphologically much like Chinese sand pears, making taxonomists unable to classify the cultivars of Chinese pears. In this study some Chinese white pear cultivars from Anhui Province, the northern border of the natural distribution of Chinese sand pears, independently clustered together into Group 4, which reflect that pear cultivars derived from the same geographic location generally had close relationships. Because they are physiologically closer to Chinese sand pears, some authors also classify them as Chinese sand pears (Pu et al., 1989). P. betulaefolia Bge. is distributed in North China (Yu, 1979) and has been extensively used for pear rootstocks in East Asia. Two clones from different places clustered together and just located between Occidental pears and other Oriental pears in dendrogram, which is consistent with the results obtained from RAPD markers (Teng et al., 2002). The position of P. betulaefolia in dendrogram resulting from both RAPD and AFLP data may indicate that it would be a transitional species between the Occidental pears and Oriental pears, as having been proposed by Challice and Westwood (1973). In summary, a high level of genetic variability among the Pyrus species and cultivars mainly native to East Asia was revealed by AFLP markers. Our results confirmed the close relationship between Chinese white pears and Chinese sand pears. Therefore, we treated the Chinese white pears as a cultivated group or an ecotype of Chinese sand pears: P. pyrifolia White Pear Group. The data in this study also suggest that the progenitor of Japanese pear might have Chinese origin. The information obtained from this study will be useful for understanding the origin and evolution of Asian pear cultivars.
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