2_nguyen_t_hai_ha_1647 (1)_2225583_20210327_021109

Biotechnology and Seedling JOURNAL OF FORESTRY SCIENCE AND TECHNOLOGY NO. 5 - 2017 11 GENETIC DIVERSITY OF Hemibagrus guttatus IN THAI NGUYEN PROVINCE BY RAPD MARKERS Nguyen Thi Hai Ha1, Bui Van Thang2, Tran Viet Vinh3, Tran Thao Van4 1,2Vietnam National University of Forestry 3,4Thai Nguyen University of Agriculture and Forestry SUMMARY Hemibagrus guttatus, which is a species of the Hemibagrus genus of Barridae, distributed in few large rivers in the mountains and only in the rapid and strong currents. Because the economic benefits from H. guttatus are very high, this is hunted in large numbers. This study evaluated genetic diversity of H. guttatus from 24 collected individuals in Thai Nguyen based on ten primers of RAPD marker. Results showed that the average genetic similarity value between the individuals studied was 69.6%, the genetic similarity was ranged from 0.57 to 0.89. The samples were divided into two groups based on the value of genetic similarity, in which the samples collected in Phu Luong district had a similarity value of 57% in one group, unlike the others with similarity value 65%. Following research creates a scientific basis and an important scientific proof to conservation and development of H. guttatus genetic resources in Thai Nguyen province. Keywords: Genetic diversity, Hemibagrus guttatus, RAPD, Thai Nguyen province. I. INTRODUCTION Hemibagrus guttatus, which is a species of the Hemibagrus genus of Barridae. In Vietnam, it is only found in the big rivers of the Northern such as Hong River, Da River, Lo River, Ma River; H. guttatus is distributed in China (Yunnan) and Laos. Due to the economic benefits of this species, the locals exploit this species in the wild without preserving its feeding and living habitat. Therefore, up to now, the number of H. guttatus in the wild is rapidly declined. For that reason, H. guttatus has been listed in the Vietnam Red List, the threat level VU A1c, d B2a, b - will be endangered, while declining by at least 20% as estimated due to habitat decline. Information on population structure is useful for the development of management strategies that will conserve the biodiversity associated with different species, sub-species, stocks and races (Turan et al., 2005). Thus, detailed knowledge of the population structure is needed for sound management and successful commercial fishing of this species. Genetic diversity and gene pools which help in adaptation and survival is considered to be a key component for conservation and management of populations (Andayani et al., 2001). Molecular markers along with the development of new statistical tools have indeed revolutionized the analytical power necessary to explore the genetic diversity, both in native populations and in captive lots. In recent years, a wide range of new molecular techniques has been explored and reported for fishes. Random amplified polymorphic DNA (RAPD) is one of such techniques which was first introduced by Williams et al. (1990). RAPD technique is the one of the most frequently used molecular methods for taxonomic and systematic analyses of various organisms and has provided important applications in fish. RAPD is a technique based on the PCR amplification of discrete regions of the genome with short oligonucleotide primers of arbitrary sequence (Welsh and McClelland, 1990; Williams et al., 1990). It utilizes single, arbitrary, decamer DNA oligonucleotide primers to amplify regions of the genome based on the polymerase chain reaction (Hadrys et al., 1992; Biotechnology and Seedling JOURNAL OF FORESTRY SCIENCE AND TECHNOLOGY NO. 5 - 2017 12 Williams et al., 1993). The characters assessed through RAPD are useful for genetic studies because they provide various types of data like taxonomic population or inheritance pattern of various organisms including fishes. The study of genetic diversity of H. guttatus is very much limited, so in the present study, this technique was applied to analyze the genetic relationship among H. guttatus populations. The objectives of this study are focused on the genetic variation of H. guttatus. That creates a scientific basis and an important scientific proof to conservation and development of H. guttatus genetic resources in Thai Nguyen province. II. RESEARCH METHODOLOGY 2.1. Materials A total of 24 fish specimens were randomly collected from 4 areas: Dai Tu district, Phu Luong district, Song Cong district and Thai Nguyen city (Table 1). All the fish specimens were kept in the iceboxes and brought to the laboratory for further study. The muscle tissues were isolated from freshly caught fishes and preserved at -20°C for further use. Table 1. List of H. guttatus samples used in this research Samples Collection area Samples Collection area NL1 Thai Nguyen city NL13 Song Cong city NL2 Thai Nguyen city NL14 Song Cong city NL3 Thai Nguyen city NL15 Song Cong city NL4 Thai Nguyen city NL16 Song Cong city NL5 Thai Nguyen city NL17 Song Cong city NL6 Thai Nguyen city NL18 Song Cong city NL7 Thai Nguyen city NL19 Dai Tu district NL8 Thai Nguyen city NL20 Dai Tu district NL9 Thai Nguyen city NL21 Dai Tu district NL10 Thai Nguyen city NL22 Dai Tu district NL11 Thai Nguyen city NL23 Phu Luong district NL12 Song Cong city NL24 Phu Luong district Table 2. List of primers used during this study Oligo name Sequence 5’-3’ OPA-07 GGA ACG GGTG OPA-09 GGG TAA CGCC OPA-11 CAA TCG CCGT OPA-20 GTT GCG ATCC OPAC-14 GTC GGT TGTC OPAH-01 TTC GCA ACCA Rm-1 CTG GGC ACGA Rm-2 TTC CGC CACC Rm-4 CCG CTA CCGA Rm-5 CCT TTC CCTC Biotechnology and Seedling JOURNAL OF FORESTRY SCIENCE AND TECHNOLOGY NO. 5 - 2017 13 In the present study, 10 commercially available RAPD primers (Table 2), which were purchased from IDT, USA, were used to initiate PCR amplifications. Primers were randomly selected on the basis of GC content and annealing temperature for RAPD-PCR amplifications. 2.2. Methods 2.2.1. Isolation of genomic DNA from fish tissue For the isolation of total genomic DNA, a modified protocol was followed using DNA isolation Kit (Norgen, Canada). UV-VIS spectrophotometer was used to check quality as well as quantity of isolated DNA. Optical densities of the DNA samples were measured at 260 nm and 280 nm and the concentration of extracted DNA was adjusted to 50 ng/µl for PCR amplification. 2.2.2. PCR amplification The reaction mixture (25 μl) for PCR was composed of H2O deion (9.5 µl), 2x PCR Master mix Solution (12.5 µl), 10 pmol/µl primer (1.0 µl), template ADN (2 µl). A negative control, without template DNA was also included in each round of reactions. After preheating for 5 minutes at 94°C, PCR was run for 35 cycles. It consisted of a 94°C denaturation step (1 min), 37°C annealing step (1 min) and 72°C elongation step (2 min) in a thermal cycler (Applied Biosystems, USA). At the end of the run, a final extension period was appended (72°C, 10 min) and then samples were stored at 4°C until the PCR products were analyzed. 2.2.3. Agarose gel electrophoresis The amplified DNA fragments were separated on 1.2% agarose gel and stained with Redsafe solution (Norgen, Canada). A low range DNA marker of 1Kb from Norgen, Canada was run with each gel. The amplified pattern was visualized on an UV transilluminator and photographed by gel documentation system (BIORAD, USA). 2.2.4. Statistical analysis The DNA fragments were scored for the presence and absence of fragments on the gel photographs and DNA fragments were compared among the H. guttatus individuals. RAPD banding patterns were recorded on spreadsheets, which were used to determine gene diversity, gene flow, number of polymorphic loci and genetic distance through a construct by an un-weighted pair group method of the arithmetic mean of UPGMA (Nei, 1978). Genetic relationships among 24 individuals were constructed by UPGMA method using NTSYS v.2.1. III. RESULTS AND DISCUSSION 3.1. Qualitative estimation of DNA The quality of DNA extracted from different H. guttatus samples were analyzed by staining with Redsafe solution and the bands were seen under UV light. The single sharp bands in all the 24 lanes clearly indicated the presence of DNA in all samples. Spectrophotometric analysis of the DNA samples showed the 260 nm/280 nm ratio of the samples obtained in range from 1.8 to 2.0 which indicates the presence of pure DNA. 3.2. RAPD polymorphic analysis The RAPD profile of bands obtained in the 24 varieties of H. guttatus with 10 random primers is shown in Table 3. These 10 random primers generated a total of 56 bands in all the Biotechnology and Seedling JOURNAL OF FORESTRY SCIENCE AND TECHNOLOGY NO. 5 - 2017 14 24 individuals out of which 66.70% bands were polymorphic. Bands size ranged from 0.3 – 2.0 Kb. Among these 10 primers, there was one primer which has polymorphism 100% (Rm-2), 6 primers had polymorphism over 50% (OPA-07, OPA-09, OPA-20, OPAH-01, Rm-1, Rm-4) and 3 primers had polymorphism less than 50% (OPA-11, OPAC-14, Rm-5). Table 3. Pattern of polymorphism of 10 random primers Primer Total No. of DNA fragments Total No. of polymorphic DNA fragments The rate of polymorphism (%) OPA-07 6 4 67.7 OPA-20 5 3 60.0 OPA-09 6 4 67.7 OPA-11 5 2 40.0 OPAC-14 6 2 33.3 Rm-5 5 3 36.0 OPAH-01 6 5 83.0 Rm-1 6 4 67.0 Rm-2 6 6 100 Rm-4 5 4 80.0 Total 56 37 66.1 The genetic similarity reflects the genetic relationship of the H. guttatus individuals. The greater is the similarity between two samples, the higher is their genetic similarity, and if there is the genetic similarity between two samples, the genetic relationship between them is closer. Results showed that the correlation coefficient between lobed tick samples ranged from 0.393 (NL13 and NL24) to 0.893 (NL19 and NL20) respectively from 39.3% to 89.3%. and the average genetic similarity value between the individuals studied was 69.6%. The results showed that the samples of H. guttatus in Thai Nguyen have a moderate genetic diversity compared to some other authors' studies using the same primers. According to Yoon et al. (2001), the polymorphic ratio of five RAPD primers including OPA09 (as used in this study) in Korean catfish (Silurus asotus) ranged from 56.4% to 59.6%. Abdul Muneer et al. (2009) have investigated the polymorphic random DNA amplification (RAPD) to investigate the patterns and distribution of genetic diversity in the natural population of tilapia in the Western Ghat-India. The study used 32 samples and 10 random primers to evaluate genetic diversity, resulted in a total of 124 bands, including 75 polymorphic bands (60.48%). The heterozygosity of 0.72 indicates a high level of genetic diversity in the studied population. Biotechnology and Seedling JOURNAL OF FORESTRY SCIENCE AND TECHNOLOGY NO. 5 - 2017 15 Table 4. Nei’s (1972) genetic similarity among 24 samples of H. guttatus estimated from RAPD profiles NL1 NL2 NL3 NL4 NL5 NL6 NL7 NL8 NL9 NL10 NL11 NL12 NL13 NL14 NL15 NL16 NL17 NL18 NL19 NL20 NL21 NL22 NL23 NL24 NL1 1.00 NL2 0.71 1.00 NL3 0.71 0.68 1.00 NL4 0.79 0.79 0.64 1.00 NL5 0.73 0.84 0.70 0.80 1.00 NL6 0.61 0.68 0.64 0.64 0.66 1.00 NL7 0.71 0.68 0.75 0.68 0.66 0.75 1.00 NL8 0.70 0.66 0.63 0.63 0.68 0.80 0.73 1.00 NL9 0.71 0.61 0.71 0.64 0.63 0.79 0.79 0.77 1.00 NL10 0.63 0.63 0.66 0.55 0.61 0.66 0.66 0.75 0.73 1.00 NL11 0.75 0.68 0.71 0.68 0.66 0.64 0.68 0.73 0.68 0.70 1.00 NL12 0.75 0.82 0.71 0.75 0.77 0.68 0.71 0.63 0.64 0.66 0.71 1.00 NL13 0.79 0.79 0.71 0.79 0.84 0.64 0.68 0.63 0.61 0.55 0.75 0.86 1.00 NL14 0.66 0.59 0.55 0.66 0.57 0.63 0.52 0.68 0.59 0.61 0.70 0.73 0.70 1.00 NL15 0.71 0.61 0.64 0.68 0.66 0.64 0.64 0.73 0.68 0.66 0.64 0.61 0.61 0.63 1.00 NL16 0.71 0.64 0.68 0.75 0.70 0.68 0.68 0.66 0.71 0.70 0.71 0.71 0.64 0.66 0.64 1.00 NL17 0.64 0.79 0.71 0.57 0.77 0.82 0.71 0.73 0.68 0.70 0.68 0.75 0.71 0.59 0.61 0.61 1.00 NL18 0.70 0.70 0.77 0.66 0.75 0.73 0.84 0.75 0.77 0.75 0.77 0.77 0.77 0.64 0.63 0.77 0.77 1.00 NL19 0.71 0.68 0.61 0.71 0.73 0.54 0.61 0.59 0.61 0.52 0.75 0.71 0.79 0.70 0.61 0.61 0.64 0.63 1.00 NL20 0.64 0.64 0.61 0.68 0.73 0.57 0.57 0.55 0.61 0.52 0.71 0.75 0.75 0.70 0.57 0.68 0.61 0.63 0.89 1.00 NL21 0.68 0.64 0.68 0.68 0.63 0.64 0.57 0.59 0.64 0.63 0.75 0.71 0.75 0.77 0.57 0.61 0.68 0.63 0.79 0.75 1.00 NL22 0.71 0.61 0.64 0.64 0.70 0.64 0.68 0.66 0.75 0.59 0.75 0.68 0.75 0.55 0.68 0.64 0.61 0.77 0.68 0.68 0.64 1.00 NL23 0.59 0.55 0.63 0.48 0.57 0.59 0.59 0.57 0.63 0.57 0.59 0.52 0.48 0.54 0.59 0.55 0.73 0.54 0.59 0.55 0.59 0.63 1.00 NL24 0.54 0.50 0.57 0.46 0.48 0.61 0.64 0.59 0.68 0.59 0.61 0.50 0.39 0.55 0.57 0.61 0.61 0.59 0.54 0.54 0.54 0.61 0.84 1.00 Biotechnology and Seedling JOURNAL OF FORESTRY SCIENCE AND TECHNOLOGY NO. 5 - 2017 16 Based on the genetic similarity between samples, NTSYS software automatically arranges samples with similarity coefficients into one group and results are shown in a systematic tree graph. Jaccard numbers and UPGMA subtypes indicate the degree of genetic variation between the 24 specimens. Different levels are expressed by the difference coefficient between the samples. Figure 1. UPGMA dendrogram on the basis of Nei’s (1972) genetic distance values at nodes represent proportion of similar replicates in H. guttatus species Figure 1 shows that 24 specimens of H. guttatus were divided into two groups with a genetic similarity 0.57. Group 1 consisted of 22 samples divided into 5 subgroups with a genetic similarity 0.65. Of which, group 1 is divided into two sub-groups: Sub-group 1 has a similarity 0.682, consisting of 9 samples: NL1, NL2, NL4, NL5, NL11, NL12, NL13, NL16, NL22; And sub-branch NL14, NL19, NL20, NL21.Sub-group 2: has a similarity 0.698, consisting of 9 samples: NL3, NL6, NL7, NL8, NL9, NL10, NL15, NL17, NL18. Group 2 contains only samples NL23 and NL24 with high genetic similarity 0.835. In conclusion, group 1 consisted of individuals which were collected in Thai Nguyen City, Song Cong City, and Dai Tu District. In particular, the individuals collected in two cities and district are scattered in both sub-groups 1 and 2, so there is a possibility of cross-genetic transfer between individuals of the two regions. Group 2 includes only individuals from Phu Luong District. Results of this analysis showed that the genetic similarity was ranged from 0.57 to 0.89. Acknowledgement Authors are thankful to the project "Conserving Hemibagrus guttatus gene resource in Thai Nguyen province" by the Thai Nguyen Science and Technology Development Fund - Department of Science and Technology Thai Nguyen province for funding for executing this research. REFERENCES 1. Abdul Munee PM, Gopalakrishnan A, (2009). Gentic variation and population structure of endemic yellow catfish, Horabagrus brachysoma among three populations of Western Ghat region using RAPD and microsatellite markers. Molecular Biology Reports, 36(7): 1779 – 1791. 2. Andayani N, Morales JC, Forstner MRJ, Supriatna J, Melnick DJ, (2001). Genetic variability in mtDNA of the silvery gibbon: implications for the conservation of a critically endangered species. Cons Biol, 15(3):1545 - 1548. 3. Duong Thuy Yen, Pham Thanh Liem, Huynh Ky and Tran Ngoc Hai, (2013). Strain evaluation of giant freshwater prawn (Macrobrachium rosenbergii) based Biotechnology and Seedling JOURNAL OF FORESTRY SCIENCE AND TECHNOLOGY NO. 5 - 2017 17 on morphology and genetic diversity. The proceedings of the International Fisheries Symposium, Agricultural Publishing House, 239-244. 4. Hadrys H, Balick M, Schierwater B, (1992). Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol Ecol, 1: 55 – 63. 5. Nei, M. (1972) Genetic distances between populations. American Naturalist, 106, 283–292. 6. Nguyen Duc Thanh, (2014). DNA marker techniques in study and selection of plant. Journal of Biology, 36(3): 265 - 294. 7. Turan C, Yalcin S, Turan F, Okur E, Akyurt I, (2005) Morphometric comparisons of African catfish, Clarias gariepinus, populations in Turkey. Folia Zoologica, 54(1-2):165–72. 8. Vietnam Red List, (2007). Natural Science and Technology Publishing House. Hanoi, 135. 9. Welsh J, McClelland M, (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res, 18: 7213 - 7218. 10. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV, (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res, 18: 6531 - 6535. 11. Williamson JH, (2001). Broodstock management for imperilled and other fishes. In G. A. Wedemeyer, minisatellite DNA single locus probes for Atlantic salmon, Salmo salar L. J Fish Biol, 37: 991 - 993. 12. Yoon JM and Kim GW, (2001). Random amplified polymorphic DNA-polymerase chain reaction analysis of two different populations of cultured Korean catfish Silurus asotus. Journal of Biosciences, 26: 641 - 647. ĐA DẠNG DI TRUYỀN LOÀI CÁ LĂNG CHẤM (Hemibagrus guttatus) TẠI THÁI NGUYÊN BẰNG CHỈ THỊ RAPD Nguyễn Thị Hải Hà1, Bùi Văn Thắng1, Trần Viết Vinh2, Trần Thảo Vân2 1 Viện Công nghệ sinh học Lâm nghiệp, Đại học Lâm Nghiệp 2 Đại học Nông Lâm Thái Nguyên TÓM TẮT Cá lăng chấm (Hemibagrus guttatus) thuộc chi Cá lăng (Hemibagrus), họ Cá lăng (Bagridae), phân bố ở các con sông lớn thuộc các tỉnh phía Bắc như ở sông Hồng, sông Đà, sông Lô, sông Mã. Cá lăng chấm có giá trị kinh tế rất cao do đó chúng bị săn bắt với số lượng lớn nên đến nay số cá thể còn lại trong tự nhiên không nhiều. Đánh giá mức độ đa dạng di truyền của 24 mẫu Cá lăng chấm thu được tại Thái Nguyên dựa trên mười chỉ thị RAPD; kết quả cho thấy mức độ tương đồng di truyền trung bình giữa các mẫu nghiên cứu 69,6%, dao động trong khoảng 0,57 đến 0,898. Các mẫu nghiên cứu chia thành hai nhóm chính dựa vào hệ số tương đồng di truyền, trong đó các mẫu thu được tại huyện Phú Lương có hệ số tương đồng 57% thuộc một nhóm, khác với các mẫu còn lại có hệ số tương đồng 65%. Kết quả nghiên cứu là cơ sở khoa học phục vụ cho việc bảo tồn và phát triển nguồn gen Cá lăng chấm tại Thái Nguyên. Từ khóa: Cá lăng chấm, đa dạng di truyền, RAPD, Thái Nguyên. Received : 22/8/2017 Revised : 08/9/2017 Accepted : 15/9/2017