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Journal of Scientific and Engineering Research, 2019, 6(10):16-23

ISSN: 2394-2630 Research Article CODEN(USA): JSERBR

Genetic Diversity and Genetic Relationship of Collected in Van Gian District, Hung Yen Province, Vietnam

Dang Van Dong1, Mai Thi Ngoan1, Phan Ngoc Diep1, Nguyen Truong Khoa2, Khuat Huu Trung2, Tran Dang Khanh2*

1Fruit and Vegetable Research Institute, Trau Quy, Gia Lam, Hanoi, Vietnam 2Agricultural Genetics Institute, North Tuliem, Hanoi, Vietnam Abstract Camellia is one of the largest genera of flowering that belongs to the family. In Vietnam, there are numerous appears to be very similar to many taxa of Camellia which is popularly used as the decorative plants with the high-value property. In this study, twelve Camelliaspecies collected from Hung Yen province were identified based on the ITS1-rRNA-ITS2 sequences. The results showed that the genetic similarity among the samples was ranged from 70.01% to 93.12%. The highest homologous coefficient was 93.12%, whereas the lowest was 70.01%, respectively. The nearest genetic distance was 0.05 and the farthest was 0.29 and divided into 2 main groups with 7 and 5 taxons based on the phylogenetic . It suggests that some Camellia samples may derive in the same origin, evolution and arising relation. This information may be useful for identifying the high-quality genetic plant sources for propagation, development and conservation of the high-valued materials of Camellia plants in Vietnam.

Keywords Camellia species, genus, genetic diversity, Theaceae Introduction Camellia is one of the largest genera of that belongs to the Theaceae family and was firstly coined by the Swedish botany Carolus Linnaeus in 1735. It is native to Southern, Eastern Asia and China which are over 80% of the species and are the center of species diversity [1]. This genus is widely grown in eastern and southern Asia including Nepal, China, Korea, Japan and Vietnam. Presently, there are approximately 100 to 300 described species and about 3000 hybrid cultivars of Camellia that is available in the world. However, presently, the interspecies relationship of the certain number of Camellia species is yet a matter argument and controversy. According to the report of Prince [2], Camellia is considered a single genus enclosing all 17 genera. The economically important species in Camelliais C. sinensis and C. assamica which are being used to make beverage [3, 4]. Some others such as C. japonica, C. reticulata, and C. sasanqua and a group of yellow flowering species narrated golden are being grown as ornamental trees and widely spread in worldwide [1]. Morphologically, Camellia is a rounded or rounded-cuneate seed with thick cotyledons, the flower can be single, semi-double flowered or double-flowered. The leaves are dark green, glossy [5]. Amongst Camellia species, especially, C. sinensis is known to contain numerous secondary metabolites which are good for human healths, for example, caffeine, xanthines, theobromine and tannins, including flavonoids, polyphenol, fats and vitamin C [6]. Recently, its consumption is increased because of its preventive effects against certain human disease such as, antiaging, anticancer, cardiovascular, anticaries, antiparkinson, antioxidant [7]. In Vietnam, there are numerous species appears to be very similar to many taxa of Camellia which was previously identified by some researchers such as C. piquetiana, C. honbaensis [8], C. dalatensis, C. longii, C. dongnaiensis, C.

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piquetiana, C. sonthaiensis etc [9, 10, 11, 12]. They are widespread to many areas from north to south in this country. Evaluation of genetic diversity and relationships of plantsare generally very important work for its breeding and classification. Some works to assess genetic diversity and population structures of C. Japonica in Japan and China were made by molecular marker [13, 14]. In Vietnam, there is sporadical research on genetic diversity of Camellia species by molecular markers. Therefore, the objective of the current study was to make a sequence database of the internal transcribed spacer ITS1- 5.8S-ITS2 region for Vietnamese Camellia species in Van Giang, Hung Yen province, Vietnam where are considered as the center of Camellia species.

Materials and Methods Plant materials A total of 12 samples species were kindly provided by the Fruit and Vegetable Research Institute in 2018. They were collected at Phung Cong commune, Van Giang district, Hung Yen province. The native name and collected areas were shown in Table 1. Table 1: List of the species used in analyses in this study No Sample name Collected areas 1 Tra luu co VN Phung Cong-Van Giang- Hung Yen 2 Tra luu co Nam Dinh Phung Cong-Van Giang- Hung Yen 3 Tra tham hong bat dien Phung Cong-Van Giang- Hung Yen 4 Tra phan bat dien Phung Cong-Van Giang- Hung Yen 5 Tra thom TQ Phung Cong-Van Giang- Hung Yen 6 Tra cung dinh VN Phung Cong-Van Giang- Hung Yen 7 Tra cung dinh do TQ Phung Cong-Van Giang- Hung Yen 8 Tra phan Japan Phung Cong-Van Giang- Hung Yen 9 Tra tham don Phung Cong-Van Giang- Hung Yen 10 Tra bach TQ Phung Cong-Van Giang- Hung Yen 11 Tra bach VN Phung Cong-Van Giang- Hung Yen 12 Tra bach nhi Phụng Công –Van Giang- Hung Yen

Total DNA extraction, PCR and DNA sequencing of the samples The fresh leaves of all samples were extracted for total genomic DNA by using CTAB method (cetyltrimethylammonium bromide) of Hoyle and Doyle [15] with some minor modifications. The yielded DNA products were then recorded by using a spectrophotometer [16]. PCRs and DNA sequencing: nuclear ribosomal Internal Transcribed Spacers including ITS1 and ITS8 with nucleotide sequence following to the method of Trung et al [17] as shown in Table 2.

Table 2: List of the primers ITS used in this study No Nucleotide sequence ITS1 5’ TCCGTAGGTGAACCTTGCGG 3’ ITS8 5’ GCACTACGATGAAGAACGCT 3’ Amplification was made in a polymerase chain reaction (PCR) in the tuber 0.2ml which contained 9.0 µl, Buffer Mg+ 25 Mm (1.5µl), dNTPs 10 Mm (0.3µl), Taq ADN polymerase 5 U/µl (0.2µl), ITS1 10 µM (1.5µl), ITS8 10 µM (1.5µl) and DNA 50ng/µl (1.0µl), respectively. Amplification reactions were conducted as shown in Table 3.

Table 3: PCR reaction procedures Step Temperature (Co) Time Cycle 1 94 5 min 1 2 94 1 min 3 58 45 s 35 4 72 50 s 5 72 7 min 1 6 4 ∞ 1

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Sequence analyses The purified PCR products were directly sequenced by an ABI PRISMTM 310 Genetic Analyzer (Applied Biosystem). The primers ITS1 and ITS8 were used for the sequence reaction. The ITS region of each individual was then sequenced in both 5’ and 3’ directions at least twice to avoid mutation introduced by Taq polymerase. The size of DNA segment was amplified by ITS 1-8 which was ranged from 700 bp to 800 bp. The boundaries of the ITS1 and ITS8 were determined by comparing them with the published sequences based on the similarity sequence on the NCBI.

Statistical Analysis The sequences were aligned and analyzed by using MEGA v5.1 program to generate the phylogeny.

Results and Discussion Molecular markers application to identify Camellia species based on ITS region sequences By application ofITS1/ITS8 primer pairs, the ITS region was successfully amplified by PCR. The product was then examined on the gel agarose (1.5%). The results were indicated to be high quality with the appearance of only one band with the length size ranged from 700- 800 bp (Figure 1). Our result was agreed with some previous reports who amplified the ITS region of the plant samples [18]. To emphasize that the obtained bands were the obvious and correct size, which are enough quality to use for sequencing.

Figure 3: Electrophoresis of amplification ITS segment on the 12 Camellia samples by PCR with primer pairs of ITS1 and ITS8

Sequencing ITS-rDNA of the Camellia samples A total of 12 PCR products which was from 12. Camellia samples was sequenced by Macrogen company (Korea). The collected data were analysed and compared by using the ClustalW of the software MEGA v6 and CLC 8.0. The results revealed the difference between the sequences which were mainly single polymorphic positions (SNP), of which one nucleotide was replaced by another nucleotide in the sequencing region of ITS1- 5.8S – ITS2 of the Camellia samples as shown in Figure 4. As a total of 12 Camellia samples were indicated the significant difference in the ITS1-5.8S – ITS2 region. The variation of the sequences between the studied samples was the first of 100 nucleotides and the last of 200 nucleotides. Based on the comparison of the nucleotides sequence. It suggested that by using of ITS1-5.8S – ITS2 which is possible to distinguish the Camellia samples. The nucleotide composition of ITS1-5.8S – ITS2 sequences of 12 samples was shown in Table 4. Some previous studies were done in Japan, Korea and China showed that the high-level diversity and nucleotide diversity in Camellia species by using molecular markers of AFLP, SSR [19, 20, 21].

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Figure 4: Comparison of nucleotide sequences of the 12 .Camellia samples As shown in Table 4, based on the ITS1-5.8S – ITS2 region, the composition of Guanin (G), Cytosine (C), Adenine (A) and Thymine (T) were the difference among the studied samples. In general, the rations of Guanin and Cytosine were shown to be higher than the Adenine and Thymine. On the other hand, the percentage of GC (%) was greater than AT (%), respectively. Particularly, Tra luu co Nam Dinhwas the highest G+ C by 67.8%, however, its A+ C was shown the lowest by 32.2%, respectively. The average percentage of G+C of all 12 Camellia samples was 64.9%, while the A+T was 35.1%, respectively. The difference in the sequence of ITS1-5.8S – ITS2 regions between the Camellia samples compared based on the similarity coefficients of each sample which was calculated by the genetic distance tool CLC v8, 02 as presented in Table 5. Table 4: The nucleotide components of the 12 Camellia samples No Sample name T(U) C A G %GC %AT 1 Tra luu co VN 15.4 33.7 19.5 31.5 65.2 34.8 2 Tra luu co Nam Dinh 13.5 35.9 18.7 31.9 67.8 32.2 3 Tra tham hong bat dien 12.6 29.6 24.1 33.7 63.3 36.7 4 Tra phan bat dien 14.8 31.8 20.7 32.7 64.5 35.5 5 Tra thom TQ 15.4 31.8 20.4 32.4 64.2 35.8 6 Tra cung dinh VN 14.3 32.8 20.9 32.0 64.8 35.2 7 Tra cung dinh do TQ 13.7 31.1 21.0 34.3 65.3 34.7 8 Tra phan Japan 14.3 31.1 21.6 33.1 64.2 35.8 9 Tra tham don 14.5 33.8 18.4 33.3 67.1 32.9 10 Tra bach TQ 13.9 34.7 18.7 32.7 67.5 32.5 11 Tra bach VN 19.8 32.5 19.0 28.8 61.3 38.7 12 Tra bach nhi 13.6 30.2 22.7 33.5 63.7 36,3 Average 14,6 32.4 20.5 32.5 64.9 35.1

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Based on the analyses of ITS1-5.8S – ITS2 sequences, it showed very genetically diverse with a high genetic similarity among the collected Camellia samples at Van Giang district, Hung Yen province. The highest homologous coefficient was 93.12%, whereas the lowest was 70.01%, respectively. The nearest genetic distance was 0.05 and the farthest was 0.29, which implies that some Camellia samples may derive in the same origin, evolution and arising relation which will be better to understand the genetic similarity and origin of the studied Camellia samples. The genetic variation occurred in the region of ITS1-5.8S – ITS2 which indicated the diversification of the collected samples areas. This information may be useful for identifying the high-quality genetic plant sources for propagation, development and conservation of the high-valued materials of Camellia plants in Vietnam. Table 5: Correlation coefficient and genetic distance of 12 .Camellia samples

The phylogenetic trees based on nucleotide sequences of ITS1-rRNA-ITS2 regions among the12Camellia samples When the region of nucleotide sequences of ITS1-rRNA-ITS2 was established, the phylogenetic trees were made by Mega 6.0 software according to the method of Maximum likelihood as shown in Figure 5. Based on the , the region of ITS1-rRNA-ITS2sequence of 12 samples were divided into 2 main groups: Group I: there were included 07 taxons as followed: Tra tham hong bat dien, Tra bach nhi, Tra cung dinh VN, Tra thom TQ, Tra phan Nhat Ban and Tra bat dien. Group II: The five taxons consisted of Tra bach TQ, Tra bach VN, Tra tham don, Tra luu co VN and Tra luu co Nam Dinh, respectively.

In conclusions, based on the ITS1-rRNA-ITS2 sequences of 12 .Camellia samples, the genetic similarity of the samples was ranged from 70.01% to 93.12%. The orthodox sequencing region of ITS1-rRNA-ITS2 of the Camellia samples was rather high and varied fluctuations on the ITS regions. The phylogenetic trees analyses and combined with the sequence similarity values of ITS1-rRNA-ITS2 regions were evaluated for accuracy sample identification in this country.

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TRA THAM HONG BAT DIEN TRA BACH NHI TRA CUNG DINH VN TRA THOM TQ TRA CUNG DINH DO TQ TRA PHAN JAPAN TRA PHAN BAT DIEN TRA BACH TQ TRA BACH VN TRA THAM DON TRA LUU CO VN TRA LUU CO NDINH

Figure 5: The phylogenetic trees of 12 Camellia samples

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