J. Sci. Dev. 2009, 7 (Eng.Iss.1): 30 - 35 HA NOI UNIVERSITY OF AGRICULTURE 30 Assessment of Genetic Variation in Local and Exotic Lilium spp. Germplasm Using RAPD Markers Đánh giá đa dạng di truyền nguồn gen Lilium spp. bản địa và nhập nội bằng chỉ thị RAPD Nguyen Thi Phuong Thao, Ninh Thi Thao, Vu Quang Khanh, Nguyen Quang Thach Faculty of Biotechnology, Hanoi University of Agriculture TÓM TẮT Ở Việt Nam, ngoài loài hoa loa kèn trắng Lilium longiflorum, các giống Lily thuộc nhóm Oriental và các con lai Asiatic đã đang trở nên rất phổ biến. Để thiết lập một chương trình chọn tạo giống trong nước đối với loài hoa có giá trị kinh tế này, việc thu thập nguồn gen với sự đa dạng di truyền lớn là yêu cầu tiên quyết. Chỉ thị RAPD đã được sử dụng để đánh giá sự đa dạng di truyền của 32 mẫu giống đại diện của các loài/ giống Lilium bản địa và nhập nội khác nhau. Trong số 20 mồi RAPD sử dụng, chỉ có 3 mồi (chiếm 15%) là các mồi cho đa hình. Ba mồi OPA10, P615 và P650 đã tạo ra được tổng số 123 băng DNA với trung bình đạt 0,8; 1,7 và 3,8 băng DNA tính trên mỗi mẫu giống nghiên cứu. Mức độ đa hình khác nhau được thể hiện bởi 3 mồi này cũng đã quan sát được ở các nhóm Lilium khác nhau. Số băng DNA đa hình cao nhất với 61 băng thu được ở nhóm chứa các giống Lily thuộc nhóm Oriental đạt 49,59%, tiếp sau đó là nhóm L. longiflorum và các con lai của chúng đạt 33,3%, nhóm các loài Lily hoang dại thu thập từ Nhật Bản đạt 8,13%; nhóm các giống lily thuộc nhóm Asiatic đạt 7,32% và nhóm các loài lily bản địa của Việt Nam đạt 1,63%. Phân tích RAPD cùng với việc thiết lập cây phân loại di truyền đã cho thấy sự đa dạng và mối quan hệ di truyền giữa các nguồn gen Lilium đã thu thập. Từ khoá: Chỉ thị DNA, đa dạng di truyền, đa hình DNA, Lilium, PCR, RAPD. SUMMARY In Vietnam, apart from Lilium longiflorum, Oriental lilies and its Asiatic hybrids have become very popular. To set up a breeding program for this high economic value crop in the country, collection of germplasm with a broad genetic variation is a prerequisite for the success. Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of genetic variation of 32 representative samples of different local and exotic Lilium species and cultivars. Of the 20 primers, only 3 primers (15%) showed to be informative primers that generated polymorphic patterns of PCR products. A total of 123 DNA bands were obtained with primers OPA10, P615 and P650 giving an average of 0.8; 1.7 and 3.8 DNA bands per plant accession respectively. Different level of genetic polymorphism as revealed by these 3 informative RAPD primers was found among different Lilium groups. The highest polymorphic DNA bands were obtained in group containing commercial Oriental Lilies with total 61 bands (49.59%), following by group of L. longiflorum and their hybrids (33,3%), group of wild species from Japan; (8.13%), group of commercial Asiatic Lilies (7.32%) and group of wild species from Vietnam (1.63%). RAPD analysis combined with the construction of phylogenetic tree revealed the genetic variation and genetic relationship between collected Lilium germplasm. Key words: DNA markers Lilium, DNA polymorphism, genetic variation, PCR, RAPD. Assessment of genetic variation in local and exotic Lilium spp. 31 1. INTRODUCTION In Vietnam, apart from Lilium longiflorum (so called "hoa loa kÌn tr¾ng" in Vietnamese), Oriental and Asiatic lilies, and their hybrids, have become very popular. Remarkably, so far Vietnam has been reported to possess three wild species which belong to genus Lilium including Lilium brownii F.E Brown, Lilium poilanei Gagn. and Lilium arboricola. Among them, L. poilanei and Lilium arboricola have been become very rare species in the world. To set up a breeding program for this high economic value crop in country, collections of germplasm with a broad genetic variation is a prerequisite for the success. Information on genetic relationship is very important in designing crop improvement program and management of germplasm. Different marker techniques (Yamagishi, 1995; Fernandez et al., 1996; Niimi et al., 1996; Roh et al 1996; Van der Meulen et al1996; Obata , et al., 2000; Marasek and Orlikowska, 2001; Abe et al., 2002) have been used for studies of genetic differentiation in Lilium populations, identification of cultivars and varieties, introgression studies, determination of parentage, phylogenetic analysis, and construction of genetic maps. Among them Random amplified polymorphic DNA (RAPD) markers is particularly well suited to high-output systems required for plant breeding because it is easy to perform, fast, reliable and of relatively low cost (Williams et al., 1990). This has been used in many studies e.g. identifying Lilium genotypes (Fern¸ndez et al., 1996; Lee et al. 1996; Choi et al., 1999; Obata et al., 2000; Yamagishi 2002); determining the hybridism of inter-specific hybrids (Yamagishi, 1995; Obata et al., 2000; Ploszaj et al. 2005; Wiejacha et al., 2001); assisting in breeding program for disease resistance (Van Heusden, 2001; detecting somaclonal variation of in vitro propagated plants (Chia-Szu Wen and Ju-Ying Hsiao. 1999; Varshney et al., 2001). The present study was undertaken to assess the genetic diversity of local and exotic germplasm of Lilium using RAPD markers and the reliable information obtained may serve as a reference for plant breeding, gene pool diversity, and germplasm conservation programs. 2. MATERIALS AND METHODS 2.1. Plant materials A total of 32 Lilium accessions were used for the study (Table 2). These were categorized into 5 major groups A: Lilium longiflorum and their hybrids; B: wild species from Vietnam; C: wild species from Japan; D: commercial Oriental Lilies and E: commercial Asiatic Lilies. 2.2. Methods RAPD-PCR: DNA was isolated from the leaves of juvenile plants growing in the Greenhouse using method of Nobuo Kobayashi (1998). In PCR-RAPD reactions, 20 primers, whose sequences are given in Table 1, were used. PCR was performed in a 20 ml reaction solution containing 10x PCR buffer, 40 ng of total DNA, 3 mM MgCl2, 200 mM each dNTP, 0.2 mM primer and 0.5 unit Taq DNA polymerase (Fermentas). PCR program was set for each primer following the instruction of references (Table 1). The amplified fragments were electrophoresed in 2% agarose gels (Quiagen, USA) followed by ethidium bromide staining. All amplification reactions were repeated at least three times. 3. RESULTS AND DICUSSION Of the 20 primers, only 3 primers (15%) showed to be informative primers that generated polymorphic patterns of PCR products. A total of 123 DNA bands were obtained with primers OPA10, P615 and P650 giving an average of 0.8; 1.7 and 3.8 DNA bands per one plant accession respectively. Figure 1. showed the example of RAPD markers generated by OPA-10. Ten-base random primers have usually been used for RAPD analysis in plant species. However, Yamagishi (1995) reported that the efficiency of 10-base primers to produce polymorphic bands was low in Lilium, i.e., 16% of the 10-base primers produced polymorphic fragments among 13 species and 16 inter-specific hybrids. In another study in 2002, Yamagishi et al. indicated that long random primers have a high efficiency to produce DNA markers in Lilium (12% of the 10-base primers amplified polymorphic fragments between the two Asiatic hybrid lily cultivars and 54 and 67% of the 15- and 20-base random primers produced polymorphic bands, respectively). In our study using different plant clones and species, 2 out of 3 informative primers were 17-base random primers accounting for 66,7 %. Long random primers might be useful in a RAPD assay for Lilium species. Nguyen Thi Phuong Thao, Ninh Thi Thao, Vu Quang Khanh, Nguyen Quang Thach 32 As seen in table 2, plant accessions were categorized into 5 major groups A, B, C, D and E. Different level of genetic polymorphism as revealed by these 3 informative RADP primers was found among different groups. The highest polymorphic DNA bands were obtained in Group D with total 61 bands (49.59 %), following by group A (33, 3 %), C (8.13 %), E (7.32 %) and B (1.63 %). However, three primers failed to produce any scorable band in individual plant of Lilium longiflorum from Gialam, Lilium poilanei Gagn. from Sapa, Lilium oriental “Crystal Star” and Lilium asiatic hybrid “Quinta” from the Netherlands and Lilium asiatic from Japan. This might suggest the distinct DNA sequences of these species. Within group A of L. longiflorum, different DNA bands were recorded indicating the genetic variation have occured and they seemed to differ from those lately imported such as Lilium longiflorum “Deliana” or Lilium longiflorum “Snow Queen”. In 2007, Trinh Thi Viet Chung also detected the morphological variations among population of L. longiflorum collected in different locals in Vietnam. This might be the result of selection and adaptation process of different clones since L. longiflorum was the earliest cultivar that has been introduced into Vietnam since 18 century. Figure 2 showed the phylogenetic dendrogram of 32 individual accessions with 3 informative primers using the program NTSYSpc 2.02h. The result showed that they can be divided in two major groups which in turn can be divided into smaller subgroups.The results of genetic analyses showed that the local and exotic Lilium using in this study is composed of genetically different clones. This would offer possibilities for identifying the germplasm collections, including material for a breeding program in the country. Table 1. Primers used in RAPD analysis, their sequence and references Primers names Nucleotid sequence 5’-3’ Primer anneal temparature ( 0 C) References OPA-01 CAGGCCCTTC 32 Anushri Varshney (2001) OPA-10 GTGATCGCAG 32 OPC-6* CAGGCCCTTC 33 (random selected) OPA-04 AATCGGGCTG 32 Pham Thi Minh Phuong (2006) OPG-10 AGGGCCGTCT 34 OPG-12 CAGCTCACGA 34 A8 GTGACGTAGG 32 Wen and Hsiao (1999) A9 GGGTAACGCC 30 C5 GATAACCGCC 34 C11 AAAGCTGCGG 34 D3 GTCGCCGTCA 32 D5 TGAGCGGACA 30 Q5 CCGCGTCTTG 32 Q6 GAGCGCCTTG 32 V10 GGACCTGCTG 36 P615 GCCGTGGACTGCAGA 50 Yamagishi et al.(2002) P617 CCCGACACCAGGTGA 50 P618 TCAGGTTATCCGCCCC 48 P623 ACGGGGTTTACCGCT 48 P650 GACACGGCCCGATAG 50 Assessment of genetic variation in local and exotic Lilium spp. 33 Table 2. List of plant materials and number of amplified products obtained with primer OPA10, P615 and P650 Number of amplified products Name of species and hybrids Origin OPA10 P615 P650 Total A Lilium longiflorum and their hybrids 2 Lilium longiflorum 1 Quang Ba, Vietnam 0 3 0 3 28 Lilium longiflorum5 Gialam, Vietnam 0 0 0 0 6 Lilium longiflorum 3 Namdinh, Vietnam 2 3 0 5 18 Lilium longiflorum 4 China 3 0 2 5 5 Lilium longiflorum 2 Japan 5 2 0 7 15 Lilium longiflorum “Deliana” Japan 0 2 1 3 29 Lilium longiflorum “Georgia” Japan 2 3 5 10 30 Lilium longiflorum “Snow Queen” Japan 0 3 2 5 3 Lilium formolongo China 0 2 1 3 Subtotal 12 18 11 41 ( 33.3 % ) B Wild species from Vietnam 4 Lilium poilanei Gagn. Sa Pa - Vietnam 0 0 0 0 25 Lilium brownii Ha Giang-Vietnam 0 2 0 2 Subtotal 0 2 0 2 (1.63 %) C Wild species from Japan 31 Lilium formosanum Japan 0 4 4 8 32 Lilium speciosum Japan 0 2 0 2 Subtotal 0 6 4 10 (8.13 %) D Commercial Oriental Lilies 1 Lilium oriental “Nostalgia” The Netherlands 4 1 0 5 7 Lilium oriental hybrid “Cassandra” Japan 0 2 1 3 8 Lilium oriental “Sorbonne” The Netherlands 0 2 1 3 9 Lilium oriental “Gradisca” The Netherlands 1 3 2 6 10 Lilium oriental “Acapulco” The Netherlands 0 2 2 4 11 Lilium hybrid “Leslie” The Netherlands 0 3 1 4 12 Lilium oriental “Tiber” The Netherlands 3 1 0 4 13 Lilium oriental hybrid “Siberia” The Netherlands 0 3 1 4 14 Lilium oriental “Stargazer” The Netherlands 0 3 4 7 16 Lilium oriental “Crystal Star” The Netherlands 0 0 0 0 19 Lilium oriental “Giacondo” The Netherlands 1 1 1 3 20 Lilium oriental “Mondriann” The Netherlands 1 1 1 3 21 Lilium oriental “Valadores” The Netherlands 3 2 1 6 22 Lilium oriental hybrid “Salinas” The Netherlands 0 0 5 5 23 Lilium oriental hybrid “Shalloon” The Netherlands 0 2 2 4 Subtotal 13 26 22 61 (49.59%) E Commercial Asiatic Lilies 17 Lilium asiatic hybrid “Royal Show” The Netherlands 0 2 1 3 26 Lilium asiatic hybrid “Freya” The Netherlands 0 2 4 6 27 Lilium asiatic hybrid “Quinta” The Netherlands 0 0 0 0 24 Lilium asiatic * Japan 0 0 0 0 Subtotal 0 4 5 9 (7.32 %) Total 25 56 42 123 Average 0.8 1.7 1.3 3.8 Nguyen Thi Phuong Thao, Ninh Thi Thao, Vu Quang Khanh, Nguyen Quang Thach 34 Fig. 1. RAPD patterns generated by primer OPA-10 . Lanes M: 1 kb size marker; 1-32: L. oriental “Nostalgia”; L. longiflorum 1;L. formolongo; L. poilanei Gagn.; L. longiflorum 2; L. longiflorum 3; L. oriental hybrid “Cassandra”; L. oriental “Sorbonne”; L. oriental - trumpet “Gradisca”; L. oriental “Acapulco”; L. hybrid “Leslie”; L. oriental “Tiber”; L. oriental hybrid “Siberia”; L. oriental “Stargazer”; L. longiflorum “Deliana”; L. oriental “Crystal Star”; L. asiatic hybrid “Royal Show” ; L. longiflorum 4; L. oriental “Giacondo”; L. oriental “Mondriann”; L. oriental “Valadores”; L. oriental hybrid “Salinas”; L. oriental hybrid “Shalloon”; L. asiatic; L. brownii; L.asiatic hybrid “Freya”; L. asiatic hybrid “Quinta”; L. longiflorum 5; L. longiflorum “Georgia”; L. longiflorum “Snow Queen”; L. formosanum;L. speciosum Fig.2. Dendrogram obtained from RAPD pattern of 27 Lilium accessions 4. CONCLUSION Obviously with the ever-increasing production of new cultivars, the importance of accurate tools for assessing the genetic background and species or cultivar genuineness has become more important. M 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 M 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 Assessment of genetic variation in local and exotic Lilium spp. 35 The RAPD technique provides a useful approach for evaluating genetic differentiation, particularly in Lilium species, which is still poorly known genetically. In this work, RAPD analysis combined with the construction of phylogenetic tree revealed the genetic variation and genetic relationship between the local and exotic lily cultivars and certain wild lily species from Vietnam. However, more efficient primers will be required to bring more reliable information which will asset the breeding program for Vietnam its self using valuable indigenous germplasm such as L. poilanei and L. aboricola. 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Assessment of genetic variation in local and exotic Lilium spp. 33 Table 2. List of plant materials and number of amplified products obtained with primer OPA10, P615 and P650 Number of amplified. construction of phylogenetic tree revealed the genetic variation and genetic relationship between collected Lilium germplasm. Key words: DNA markers Lilium, DNA polymorphism, genetic variation, PCR, RAPD. . in Lilium populations, identification of cultivars and varieties, introgression studies, determination of parentage, phylogenetic analysis, and construction of genetic maps. Among them Random