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J. Sci. Dev. 2009, 7 (Eng.Iss.1): 22 - 29 HA NOI UNIVERSITY OF AGRICULTURE Genetic Variation of Wakegi Onion (Alllium Araki) in Vietnam  wakegi Đa dạng di truyền các mẫu giống hành wakegi thu thập từ Việt Nam Pham Thi Minh Phuong1, Shiro Isshiki2 and Yosuke Tashiro2 1 Department of Vegetables, Fruits, Flowers and Ornamental plants, Faculty of Agronomy, Hanoi University of Agriculture, Gialam, Hanoi, Vietnam 2 Faculty of Agriculture, Saga University, Saga city, Japan TÓM TẮT Để đánh giá nguồn gen các mẫu giống hành wakegi (Alllium × wakegi Araki) trong việc lai tạo các cây họ hành thì đa dạng di truyền trong và giữa các mẫu giống hành wakegi thu thập từ miền Bắc, Trung và Nam của Việt Nam và hành hoa, hành củ thu thập từ Nhật, Thái Lan đã được nghiên cứu dựa trên các đặc điểm hình thái, sinh lý và tính đa hình DNA của nhân, tế bào chất và ti thể. Kết quả nghiên cứu tế bào và phân tích GISH chứng tỏ tất cả các mẫu giống hành wakegi từ Việt Nam là con lai giữa hành hoa và hành củ và có tế bào chất được nhận từ hành hoa. Chúng có 8 nhiễm sắc thể nhận từ hành hoa và 8 nhiễm sắc thể nhận từ hành củ. Kết quả phân tích RAPD cho thấy 16 trong số 40 mồi sử dụng đã tạo ra 160 đoạn DNA trong tất cả các mẫu giống, trong đó có 135 đoạn đa hình. Trên cây di truyền được xây dựng từ kết quả của phân tích RAPD, sự đa dạng di truyền được thấy giữa các mẫu giống hành wakegi. Một số nhóm phụ được hình thành tuy nhiên các mẫu giống được thu từ một địa điểm không cùng vào một nhóm. Các phân tích RAPD khác loài đã thu được 145 đoạn DNA. Trong số các đoạn DNA thu được từ các mẫu giống hành wakegi thì 85 đoạn giống hành hoa và 71 đoạn giống hành củ. Trên cây di truyền 3 nhóm này được tách biệt rõ ràng lần lượt là nhóm các mẫu giống hành wakegi, nhóm các mẫu giống hành củ và nhóm mẫu giống hành hoa. Khoảng cách di truyền trên cây di truyên giữa hành wakegi và hành củ là 0.3 và giữa hành wakegi và hành lá là 0.5. Key words: Alllium × wakegi Araki, đa dạng di truyền, DNA tế bào chất, DNA ti thể, hành wakegi, RAPD. SUMMARY To evaluate wakegi onion (Alllium × wakegi Araki) strains as genetic resource for breeding of onions in Vietnam, genetic variation among and between wakegi onions collected from the North, Central and South regions in Vietnam and Japanese bunching onion, added shallot strains from Japan, Thailand were studied basing on morphological, physiological characters and polymorphisms of nuclear, chloroplast and mitochondrial DNAs. The results from cytological and genomic in situ hybridization (GISH) analyses demonstrated that all the examined wakegi onion strains from Vietnam were hybrid between Japanese bunching onion and shallot with the cytoplasm from Japanese bunching onion and possessed eight chromosomes from shallot and eight chromosomes from Japanese bunching onion in the somatic root tip cells. The intraspecific analysis among wakegi onion showed that 16 out of 40 primers used in RAPD analysis produced total 160 distinct bands in all the strains examined and 135 bands were polymorphic. In the dendrogram of RAPD data, variation was detected between wakegi onion strains. There were many sub- groups however all the strains from the same region were not always formed into one sub-group. The interspecific analysis produced total 145 DNA bands and among wakegi onion band patterns detected, 58 bands were similar to those obtained from Japanese bunching onion, 71 bands similar to those from shallot. Three distinct groups were found as shallot group, wakegi onion group and Japanese bunching onion group. The genetic distance value between wakegi onion and shallot in phylogenetic tree was 0.3, and that between wakegi onion and Japanese bunching onion was 0.5. 22 Journal of Science and Development 2009: Tập VI, No 6: 46-55 HA NOI UNIVERSITY OF AGRICULTURE Key words: Alllium × wakegi Araki, chlotoplast DNA, genetic variation, mitochondrial DNA, RAPD, wakegi onion. 1. INTRODUCTION Wakegi onion is an interspecific hybrid of Japanese bunching onion × shallot (Tashiro, 1980; Tashiro et al., 1990) and has been cultivated in Japan, China, Korea and South East Asia (Tashiro et al.,1982; Tashiro, 1984; Inden and Asahira, 1990, Buijisen, 1990; Brewter, 1994; Tashiro et al 2006). It has the cytoplasm derived from Japanese bunching onion (Tashiro,1980). Wakegi onion is another small-bulb onion with slender leaves and divides freely to form many side shoots. Unlike Japanese bunching onion, it forms bulbs under long day and enters dormancy during summer. It is not very cold – hardy and it is grown in the warmer regions of Japan (Brewster, 1994). Arifin (Arifin and Okubo, 1996; Arifin et al., 2000) reported that there was high genetic variation exiting among wakegi onion strains collected from Indonesia as revealed by isozyme, RAPD and PCR-RFLP analyses. Wakegi onion is widely cultivated in Vietnam as a winter crop. The bulbs are planted in the autumn field then the shoots grown quickly and form many tillers. However, the long dormant stage during summer and growth retardation in winter prevents this crop from year round harvesting and shipping. So far, Tashiro et al. (Tashiro et al. 2006c) had succeed to overcome the obstacles to the harvesting and shipping of wakegi onion through the plant breeding method. By doubling chromosomes of wakegi onion in callus culture, they got amphidiploid plants (2n=32), which then were crossed with Japanese bunching onion (2n=16) to raise new cultivar named “Saga –otome”. This new cultivar is triploid plant (2n=24) and propagated by bulblets. It shows light dormancy and heat tolerance, so suits to summer cultivation. In Vietnam, all the parts of wakegi onion are used as green salad, herb or to flavour the dishes (Pham, 2002). However, wakegi onion has not been distinguished from shallot in the market (Pham, 2006a) though the farmers planted them separately in the fileds. In order to clarify the origin of wakegi onion collection from Vietnam and to evaluate Vietnamese wakegi onion strains as genetic resource for future breeding of onions, genetic variation among wakegi onion strains collected from the North, Central and South regions in Vietnam was studied basing on morphological and physiological characters and polymorphisms of nuclear, chloroplast and mitochondrial DNAs. 2. MATERIALS AND METHODS 2.1. Plant materials Twenty two strains of wakegi onion were collected from the North, Central and South regions in Vietnam during trip in December 2001. The detail information on cultivated place and condition of each strain was collected by an interviewing. All the strains collected had been cultivated in the collection sites or the nearby places. These strains were cultivated in a plastic house of Saga University from September 15, 2002 to May 1, 2003 (The roof of the plastic house was covered with plastic film to protect from rainfall and frost, but both sides and doors were opened). Ten plants of each strain were used for morphological and physiological observations. 20 strains of wakegi onion from Vietnam and one additional strain from Tiengiang province (South region) were used for analysing interspecific variation via RAPD method. All the wakegi onion and two addition strains (one shallot and one Japanese bunching onion) were used in chloroplast and mitochondrial DNA analyses. Furthermore, 10 strains of Japanese bunching onion, 7 strains of shallot and 6 strains of wakegi onion were used for inter-specific analysing via RAPD method among them, Kujyo, 18-5 and XY31 were from Japan, Thailand and Japan, respectively and the rest was Vietnam. 2.2. Cytological analysis Method of cytological analysis was as the same as described by Pham T.M.P (Pham, T. M. P et al. 2006). 2.3. GISH analysis 23 Pham Thi Minh Phuong, Shiro Isshiki and Yosuke Tashiro Nuclear genome of wakegi onion accessions had been analyzed by using genomic in situ hybridization technique with a little modification. Root tips of the plants material were collected and treated with 2nM 8-hydroxyquinoline at 4oC for 24 hr. After fixation in the mixture of acetic acid and ethyl alcohol (1:3), the root tips were treated with 0.1% cellulase – onozuka RS (Yakult Pharmaceutial Co., Ltd., Tokyo), 0.1% pectolyase Y-23 (Sheishin Pharmaceutical Co., Ltd., Tokyo) and 0.1% cytohelicase in 10mM citrate buffer (pH 4.5) at 37oC for 1hr, washed in distilled water, and squashed in 45% acetic acid. After removal of the coverslips by the dry-ice method, the preparations were treated with 45% acetic acid to dehydrate the chromosomes, and the preparations were subsequently air-dried over night. Total DNA of Allium fistulosum (Kujyo) extracted by CTAB method was labeled with biotin-16-dUTP by nicktranslation Kit (Boheringer Mannheim Co. Ltd.) and used as a probe. Total DNA of shallot was used as a blocking DNA. The chromosome preparations were denaturated in 70% formamide, 2 × SSC at 70ºC for 3min, dehydrated through a prefreezed ethanol series of 70, 90 and 100% for 5 min each, and air – dried. The hybridization mixture for 100 µl (total volume) consisted of 50% formamide, 2 ×SSC, 10% dextran sulphate, 1µg of biotinylated A. fistulosum total DNA, and 30 µg of unlabeled shallot total DNA. The mixture (10 µl) was applied to each slide and covered with a plastic cover slip. Biotin- labelled DNA was detected with FITC- conjugated avidin. Each slide was treated with 50 ml of a blocking buffer (4 × SSC containing 10 mM MgSO4, 0.1% Tween 20, 3% bovine serum albumin) for 15 min at 37o C, and incubated with 50 ml of 20 mg/ml fluorescein avidin DN (Vector Laboratories, Burlingame, Calf.) in the blocking buffer for 45 min at 37oC. After washing in a washing buffer (4 × SSC containing 10 mM MgSO4, 0.1% Tween 20) five times, 3 min each, at room temperature, the signals from biotin were amplified. Each slide was incubated with 50 ml of 20 mg/ml FITCconjugated anti - avidin D (Vector Laboratories, Burlingame, Calf.) in the blocking buffer for 15 min at 37o C and then washed as described above. Chromosomes were counterstained with 1 µg/ml propidium iodide. Slides were mounted with antifade (Vectashield, Vector Laboratories Burlingame, Calf.). 24 The GISH image was observed on fluorescence microscope DMRXA (Leica Co., LTD.) using software QFISH (Leica Co., Ltd.). 2.4. Morphological and physiological observations Three characters as plant height, leaf diameter and number of tillers per plant were recorded at 2.5 months after planting. Leaf color was observed in all the growing cycle, and bulb skin color was noted after harvest. Number of days from planting to bolting was counted, and flower morphology was observed at full bloom. 2.5. RAPD analysis Method of RAPD analysis was the same as described by Pham T.M.P (Pham, T. M. P et al. 2006). 2.6. Mitochondrial DNA analysis Method of mitochondrial analysis was as the same as describled by Pham T.M.P (Pham, T. M. P et al. 2006). 3. RESULTS 3.1. Cytological analysis The results from root tip cell observations by squash and GISH methods showed that all the strains collected possessed 16 chromosomes in the root tip cell. Two satellite chromosomes were distinguished clearly; one was typical type of shallot and the other was typical type of Japanese bunching onion. In GISH analysis, 8 chromosomes from shallot were observed clearly in yellow green color and 8 chromosomes from Japanese bunching onion in orange red color (Figure 1). Among the wakegi onion examined, there was no variation in the size and shape of the chromosome except the satellite size and position. 3.2. Morphological and physiological observations Morphological and physiological characters of all the strains observed were given in table 1. All the wakegi onion strains collected had dark green and erect leaves. There were variations in all the characters observed: time for sprouting Genetic variation of wakegi onion (Alllium × wakegi Araki) in Vietnam arranged from 3.7 to 9.6 days, plant height from 4.2 to 58.8 cm, leaf diameter from 7.6 to 10.6 mm and the number of tillers per plant from 8.0 to 18.2. All the strains collected produced flower stalks in Dec.2003. The flower bud and the flower morphology were intermediate between that of shallot and Japanese bunching onion. Fig. 1. Somatic chromosomes (2n) of wakegi onion. a: Feulgen method; b: by genomic in situ hybridization (GISH) method. The red (dark) arrowheads point the satellite carried chromosomes. Yellowish green fluoresces: chromosomes of Japanese bunching onion, orange-red fluorescence: chromosomes of shallot (white arrowheads) Table 1. Morphological and physiological characteristics of wakegi onion Strain number W- 2 Collection market Plant height (cm) Leaf diameter (mm) Number of tillers per plant 5.6 ± 0.5 50.4±1.1 9.7±0.3 W- 4 " 5.2 ± 0.7 47.6±1.0 9.5±0.2 9.5±1.0 W- 5 " 7.5 ± 0.5 44.7±1.7 9.1±0.5 10.5±0.8 8.1 ± 1.1 49.3±1.8 8.9±0.3 13.2±1.1 8.0±1.1 W- 6 Baoyen, Laocai Time for sprouting (days) Sapa, Laocai 9.4±1.6 W- 7 " 9.6 ± 0.8 42.7±0.8 9.7±0.3 W- 8 " 6.0 ± 1.0 43.8±0.8 9.8±0.1 9.9±2.1 9.6±0.4 17.7±1.2 W- 24 Sapa, Laocai 4.8 ± 0.6 54.6±2.1 W- 21 Cocleu, Laocai 3.7 ± 0.2 47.9±2.4 9.0±0.3 10.3±0.1 W- 22 " 7.7 ± 0.4 42.4±0.5 10.0±0.4 10.6±0.7 W- 26 " 8.6 ± 0.5 45.0±1.8 10.1±0.3 9.2±1.6 W- 29 " 5.0 ± 0.3 48.6±4.8 9.2±0.1 17.2±1.4 W- 31 " 8.8 ± 0.3 45.4±1.9 9.3±0.5 14.7±0.3 W- 32 " 5.8 ± 0.4 48.6±1.7 9.4±0.2 18.2±1.1 W- 34 " 5.1 ± 0.4 50.7±2.6 9.4±0.1 13.6±1.0 W- 77 " 8.6 ± 0.4 47.7±1.3 9.4±0.1 12.2±0.1 W- 76 " 6.4 ± 0.4 53.1±1.3 9.0±0.2 17.8±1.6 W- 28 Huongcanh, Vinhphuc 5.5 ± 0.3 49.3±0.7 9.9±0.2 18.0±2.3 25 Pham Thi Minh Phuong, Shiro Isshiki and Yosuke Tashiro W- 30 4.8 ± 0.1 48.5±0.6 9.9±0.2 14.1±1.8 W- 44 Dongxuan, Hanoi " 8.5 ± 0.6 50.7±0.7 7.6±0.4 17.6±1.0 W- 35 Trangbang,Tayninh 5.5 ± 0.5 51.0±.14 9.7±0.2 16.7±2.0 W- 43 Dongba, Hue 6.8 ± 1.8 58.8±1.5 9.2±0.1 15.4±2.1 Table 2. Number of total and polymorphic bands detected in RAPD analysis of wakegi onion Primer Sequence No. of total bands No. of polym orphic bands OPA-02 OPA-04 OPA-10 OPA-11 OPA-16 OPA-19 OPA-20 OPG-02 OPG-03 OPG-04 OPG-06 OPG-09 OPG-11 OPG-12 OPG-13 OPG-18 Total 5’-TGCCGAGCTG -3’ AATCGGGCTG GTGATCGCAG CAATCGCCGT AGCCAGCGAA CAAACGTCGG GTTGCGATCC GGCACTGAGC GAGCCCTCCA AGCGTGTCTG GTGCCTAACC CTGACGTCAC TGCCCGTCGT CAGCTCACGA CTCTCCGCCA GGCTCATGTG 14 9 8 13 9 16 13 2 11 8 9 10 4 13 10 11 160 13 7 5 10 9 15 13 2 9 8 7 9 4 10 9 5 135 Fig. 2. Dendrogram from RAPD data 3.3. RAPD analysis In the intraspecific analysis, 16 out of 40 primers used in RAPD analysis produced total 160 distinct bands in all the strains examined (Table 2). 135 polymorphic bands were scored among the strains. In the RAPD amplification of the all the strains examined from three primers OPA-16, OPG02 and OPG-04, all the bands detected were polymorphic bands. Small variations in the presence 26 Fig. 3. Dendrogram based on RAPD data or absence of the bands could be observed between wakegi onion strains from both Vietnam and other countries. Significant variation observed between wakegi onion strains and shallot and Japanese bunching onion. In the dendrogram of RAPD data (Figure 2), variation was detected between wakegi onion strains. There were many sub- groups however all the strains from the same region were not always formed into one sub-group. In the interspecific analysis, total 145 DNA bands were detected. Genetic variation of wakegi onion (Alllium × wakegi Araki) in Vietnam Among wakegi onion band patterns detected, 58 bands were similar to Japanese bunching onion while 71 of those similar to shallot (Figure 4). From the dendrogram based on RAPD data (Figure 3), three distinct groups were found as shallot group, wakegi onion group and Japanese bunching onion group. The genetic distance value between shallot and wakegi onion was 0.3, and that between shallot and Japanese bunching onion was 0.5. Some small variations could be seen inside each group. Fig. 4. RAPD profiles generated by primer OPG -11 in Japanese bunching onion (M: Marker II; 1: Kyujo, 2 : F-25, 3: F-9, 4: F-194, 5: F-54, 6: F-126, 7: F-01, 8: F-38, 9: F-57 and 10: F-191), shallot (11: 18-5, 12: A-13, 13: A-23, 14: A-10, 15: A-52, 16: A-42, 17: A-68) and wakegi onion (18: XY3-1, 19: W-22, 20: W-28, 21: W-43, 22: W-35, and 23: W-192) 3.4. Chloroplast DNA analysis The sizes of the amplified regions were about 3.2 kbp and 4.5 kp for rbcL- ORF106 and rpoC1rpoC2, respectively in all the strains of wakegi onion. Total 27 bands were detected by digesting amplified region of rbcL-ORF106 with restriction enzymes (Alu I, Taq I, Ase I, Ecor V, Bgl II and BamH I) and 10 bands by digesting amplified product of rpoC1 - rpoC2 with Alu I. All the wakegi onion strains had the same band patterns as those of Japanese bunching onion. There was no polymorphism among the wakegi onion strains examined. 13 and 3 polymorphic bands were detected between wakegi onion and shallot in rbcL-ORF106 and rpoC1- rpoC2 regions, respectively. 3.5. Mitochondrial DNA analysis The size of the amplified region was 550 bp. Total 6 bands were observed in all the strains examined. All the wakegi onion strains had the same band patterns as those of Japanese bunching onion. Two polymorphic bands were detected between wakegi onion strains and shallot strains by digesting the amplified product with Rsa I. 4. DISCUSSION The result from cytological and GISH analyses demonstrated that all Vietnamese wakegi onion strains examined were possessed eight chromosomes from shallot and eight chromosomes from Japanese bunching onion in the somatic root tip cells. The results agreed with the formula as K(2n)=14V+J(t)1+J(T) 2 given by Tashiro (1984). The morphological observation showed that flower bud and flower of all the 27 Pham Thi Minh Phuong, Shiro Isshiki and Yosuke Tashiro strains collected were intermediate between those of shallot and Japanese bunching. Beside that, the results of the morphological and physiological observations and DNA analyses showed small variation among wakegi onion examined, but there was no variation between the strains from different regions as those of shallot and rakkyo (Pham, 2006). In DNA analyses of chloroplast and mitochondrial DNAs, all the wakegi onion had the same band patterns as those of Japanese bunching onion. All above results prove that wakegi onion in Vietnam was a hybrid between shallot and Japanese bunching onion. These results also agree with the conclusion given by Tashiro (1984) that the cytoplasm of wakegi onion was derived from Japanese bunching onion. Arifin (2000) had reported that there were shallot strains collected from Indonesia, of which cytoplast was derived from shallot from Indonesia. However from cytological analysis, one of the material plants of which cytoplasm was reported to derive from shallot, was confirmed to be aneuploid with number of chromosomes 25 (Endang, 2002). So far there was no information about bolting of wakegi onion in the South East Asia (Buijsen, J.R.M, 1990) and Vietnam (Pham, 2003). However, all the strains collected showed bolting under condition in Saga, Japan. This result proves that the wakegi onion in Vietnam has a potential to flower and that the growing condition has a remarkable effect to the flowering. The bolting of wakegi onion is the same as in the case of shallot, the short day length and/ or low temperature during autumn and winter supposed to stimulate the bolting of both types of wakegi onion under condition in Saga. Wakegi onion is a natural hybrid between shallot and Japanese bunching onion, and it has intermediate in morphological characters from both. However, in Vietnam, only one name was used for both wakegi onion and shallot (Pham, 2003; Pham, 2006b) even though the farmers could separate them in the field. The confusion between wakegi onion and shallot also happens in Indonesia (Arifin et al., 2000) and it seems to happen in the other South East Asian countries where both wakegi onion and shallot are cultivated. In Vietnam, wakegi onion is widely used during winter when Japanese bunching onion absent in the market. The culture of using green plant of Japanese bunching onion (during summer time) and wakegi onion (during winter time) since 28 history proves the importance of both species the daily life in Vietnam, therefore further study is nessesary to improve both of these species. REFERENCES Arifin, N. S., Y. and H. Okubo (1996). 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