1.                  Introduction

Lacquer is a special natural product in Southeastern Asia and Eastern Asia that was considered to be coating materials used in mankind culture for thousand years. In Asian countries, lacquer sap is wildly obtained from several species; Chinese lacquer (Toxicodendronvernicifluum(Stokes) F. Barkley) in China, Japan and Korea, wax tree (T. succedaneum (L.) Kuntze)  in Vietnam and Taiwan, Burmese lacquer (G. usitata) in Myanmar and Thailand [2] and Laccifera lacquer (G. laccifera) in Thailand and Cambodia [3, 4]. Lacquer sap is a raw material to use for many lacquer works, such like; mother-of –pear inlaid, colored glass inlay, traditional Thai painting (Lai Rot Nam, Lai Kammalor), varnish for headgears (Khon mask, crown, tiara, etc.), military armors, etc.[5,6].Historically, the oldest archaeological evidence of lacquer utilization in Thailand was found on log coffins at Pang Mapha district, Mae Hong Son Province which could be dating with range 1,960±30 years to 1,636±44 years [7]. During AD 1864-1865(early Bangkok period), lacquer sap was recorded for annual gift tribute account on lacquer sap that was submitted from MeaungUdongmeechai (Cambodia) to Siam [8] that indicated lacquer sap very important for lacquer ware handicraft. In 1959, Thailand had recorded to export lacquer exuded to Japan an amount ex exceeded 100 -150 tons [9]. The average volume of lacquer sap used in the country before 1989 at 8 tons [10]. In 2015, the lacquer sap production in Thailand was estimated less than 500 kg/year. However, it has decrease continuously after Thailand logging ban in 1989, and has import from neighboring countries now. Because form the past to present, lacquer sap was tapped from natural forest and did not establish the lacquer tree and no promotion to industry scale. As of today, the lacquer tree habitats have been loss by anthropogenic activities. Mostly, the lacquer trees have remained in protected areas, while in land with a holding has been effective by changing of land ownership[10].

The species diversity of the genus Glutain the world has about 30 species[11]while has recorded 34 accepted species in [12], its species number has recorded11 species in Thailand, namely; Glutacambodiana Pierre, G. compactaEvrard, G. elegans(Wall.) Hook.f., G. glabra (Wall.) Ding Hou, G. laccifera(Pierre) Ding Hou, G. obovataCraib, G. renghasL., G. tavoyanaWall. ex Hook.f., G. usitata(Wall.) Ding Hou, G. velutinaBlume and G. wrayiKing [13].  Glutaglabrais widely distribution more than others species, mainly in northeastern part, while Glutausitatais very important for lacquer ware industry, which has limited in northern part of Thailand up to Myanmar. Glutalacciferais scattered distributed in some areas of northeastern and upper peninsula Thailand extending to northern Cambodia[10]. The other Gluta species are a little population in the natural forests with conservation status rare species.

Molecular markers have proven to be invaluable tools for assessing plants’ genetic resources by improving our understanding with regards to the distribution and the extent of genetic variation within and among species. Recently developed marker technologies allow the uncovering of the extent of the genetic variation in an unprecedented way through increased coverage of the genome[14]. [15] reported a simple and novel DNA marker technique Start Codon Targeted (SCoT) polymorphism. It uses 18-mer single primer in PCR and PCR products are resolved using standard agarose gel electrophoresis. The primers are very easy to design based on the conserved region surrounding the translationinitiation codon, ATG[16, 17] without needing the genomic sequence information. The SCoT marker system is useful for identification and genetic diversity and variation analysis in agricultural and forestry plant speciessuch like; mango [1, 18, 19], wax tree (Rhus succedaneaL.)[20] grape [21], orchid genus Eria[22], Dendrobium nobile[23] and Diospyros species [24], etc.The objective of this study was to estimate genetic diversity among species and clones in northeastern Thailand using SCoT markers. Rich genetic diversity within and among population thus provides an important basis for maintaining their wisdom and enabling sustainable lacquer sap tapping.In view of the importance of lacquer tree in Thailand and Worldwide, this study aimed at characterizing the genetic diversity of 71 Glutaclones of northeastern Thailand using SCoTmarkers polymorphism, and to evaluate the usefulness of SCoT markers for germplasm management.

2.                  Materials and Methods

2.1.              Plant Material

A total of 3 Gluta species were selected for the present study consisting of 1 clone of Glutacambodiana, 13 clones of G. lacciferaand 57 clones of G. glabra (Table 1).  The experimental set consisted of71 samples of Glutaspecies from three biogeographically areas in northeastern Thailand ; upper northeastern (UNT)  consisting of 19 clones from Udon Thani (A6, A29 – A44, A60, A61) 2 clones from Sakorn Nakhon (A3, A4), 6 clones from Kalasin (A2, A5, A45, A46, A47, A48), 7 clones fromKhonKhaen (A62, A63, A64, A65, A66, A67, A68), western northeastern (WNT)  consisting of 23 clones from Chaiyaphum (A1, A7 –A28) and lower northeastern(LNT) consisting of 11 clones from Nakhon Ratchasima (A49 –A59) and 3 clones from UbolRatchathani (A69, A70, B1) (Table 1).Young and healthy leaf samples of Glutaspecies were collected from natural forests in 3 biogeographically areas in northeastern Thailand.

2.2.              DNA Extraction

From each sample, 0.5 g of young and healthy leaf tissue was collected and was either immediately used for DNA extraction, or stored in silica gel until completely dry before DNA isolation. Genomic DNA was isolated from the leaf tissues according to the CTAB method described by [25] with minor modifications [26]. DNA samples were stored at -20^°C and the quality verified by electrophoresis on ethidium bromide stained 1% agarose gel.

2.3.              SCoTAnalysis

All SCoT primers were synthesized by Bio Design Co Ltd.(Pathumthani, Thailand). Fouty-five SCoT primers were initially screened, using 1clone of each species, for polymorphism and reproducibility.  Each 20 μl amplification reaction consisted of 2 μl X 10X polymerase chain reaction (PCR) buffer (with 25 mM MgCl2), 0.4 μl dNTP (10 mM), 1 μl of each primer (10 μM), 0.2 μl My TagäDNA polymerase, 15.4 μl distilled water, and 1 μl cDNA template (20 ng) (Luo et al., 2010). My TagäDNA polymerase and buffer were purchased from Bio Line (Bioline Regents Ltd, USA). The procedure for the PCR reaction using a thermo cycler  C1000 (Bio-Rad, USA) include an initial denaturation step at 94°C for 3 min, followed by 36 cycles of 94°C for 50 s, 50°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 5 min. The PCR amplifications for all primers were processed using the same procedure, and all amplified products were resolved on 1.5% agarose electrophoresis in 1% Tris-Borate/ EDTA buffer and stained with ethidium bromide (0.5 μg/ml). The banding pattern images were acquired under UV light using a SynGene Gel-Doc Imaging System (SYNGENE, USA).

2.4.              Data Analysis

The band patterns obtained by each SCoT primer were scored as absent (0) or present (1). Only clear, reproducible bands were scored. Genetic similarity among clone was evaluated by calculating the Jaccard’s similarity coefficient and cluster analysis was performed using the UPGMA (Unweighted Pair Group Method of Arithmetic Means) Algorithm using NTSYS-PC software package [27].A dendrogram was produced from the Jaccard’s similarity coefficient matrices for each marker type to investigate relationships among genotypes.

3.                  Results and Discussion

3.1.              Polymorphism Detected UsingSCoTMarkers

The SCoT primers were screened according to[1]. Of the 45 primers (Primer ID 37- 80) tested in the present study, 10 primers were selected for geneticrelationship and diversity analysiswith allele size ranging from a maximum of 3,000bp (SCoT43 and SCoT48) to a minimum of 400 bp (SCoT47 and SCoT55).Among the 71clones, 10SCoT primers generated 53 bands with an average of 5.30 bands per primer ranging from 4 (SCoT54, SCoT58, SCoT63, SCoT67) to 8 (SCoT43) per primer, of which 37 were polymorphic, yielding a polymorphism rate of 69% (Table 2).  The number of polymorphic bands varied from 2 (SCoT61 and SCoT67) to 6 (SCoT43 and SCoT55) (Figure 1), with an average of 3.7 bands per primer (Table 2). The primer SCoT55 amplified the highest percentage of polymorphic bands (86%) (Table 2). No single SCoT primer could distinguish all the clones independently. These results indicate that SCoT markers could detect high polymorphism among Gluta clones. These results indicated that a high polymorphism could be detected among Glutaclones using SCoT markers. All the primers which used in this study have been performed three times on different days and using different thermal cycles, no new bands were detected.

3.2.              Genetic Diversity Analysis Among the Tested GlutaClones

Genetic diversity revealed by SCoT markers All the 53 scored bands were used to calculate genetic similarity among the 1 clone of G. cambodiana, 13 clones of G. lacciferaand 57 clones of G. glabra. Genetic similarity coefficient between pairs of accessions was obtained from the markers data based on Jaccard’s similarity coefficient.  Pairwise comparison of accessions indicated relative genetic similarity between clone ranging from a maximum of 0.97 to a minimum of 0.36 with an average of 0.67(Figure 2). The observed highest genetic similarity value (0.97) was found in clone ID No. A55 &A56ofG. laccifera,the observed lowest genetic similarity value (0.36) was found in clone ID No. B1 of G. cambodiana. The clones of G. glabrashowed the widest of the genetic similarity 0.57and 0.953,as a result of the highest of sample number (Figure 2).

The similarity coefficients generated from the SCoT data were used to construct a dendrogram based on the simple matching Jaccard’s similarity coefficient. In the present studies, the dendrogram grouped the 71 clones into 5clusters (Figure 2) at a similarity index of 0.61. The cluster ‘1’ consisted of 66 clones, which belonged to2 species (53 clones of G. glabra and 13 clones of G. laccifera).  This cluster is subdivided into 2 groups at the coefficient 0.637.The first subgroup ‘1A’ consisted of 33clones of G. glabra which originated from upper northeastern and western northeastern Thailand and the second subgroup ‘1B’consisted of 20clones of G. glabra which originated from upper northeastern Thailandand 13 clones of G. lacciferawhich originated from lower northeastern Thailand. The cluster ‘2’ consisted of only 1clone of G. glabra (A36) which originated from upper northeastern Thailand (Udonthani). The cluster ‘3’ consisted of only 1 clone of G. glabra (A7) which originated from western- northeastern Thailand (Chaiyaphum). The cluster ‘4’ consisted of 2 clones of G. glabra (A9, A11) which originated from western- northeastern Thailand(Chaiyaphum).The cluster ‘5’ consisted of 1 clone of Glutacambodianawhich was classified clearlybased on both its fruit morphological characteristicswithout fruit wing and genotype.

Assessment of genetic diversity among and within plant species is important for breeding and genetic resource conservation programs. To assess the genetic diversity and variation  in lacquer species were reported only in the genus Toxicodendron, such like,[20]reported in 96 samples of  wax tree in Thailand using SCoT primers indicated that was classified into 10 groups  with genetic variation was high and [28] reported in 109 cultivars of Chinese lacquer belonging to  five different lacquer cultivars (Damu, Dahongpao, Gaobazhi, Huangrongguizhou and Huangmao) in China analyzed by SSR markers indicated that Gaobazhi cultivar had greater genetic diversity and Gaobazhi cultivar was closer to the group containing the Dahongpao and Huangrongguizhou cultivars than Damu and Huangmao cultivars.[29] had attempted to determinate of genetic relationships among Rhus succedanea(synonym of Toxicodendronsuucedaneum) in Japanby 12 microsatellite markers that showed with numbers of alleles per locus ranging from 3 to 19 and expected heterozygosity ranging from 0.22 to 0.85.  While [30] combined molecular method of ISSR, AFLPanalysis and RAPD markersconcerning the superior candidate individuals of Rhus succedaneafor clone identification and genetic relationship among clone.

[31] reported the genus Glutawas closely to mango (genus Mangifera), it was classified in the tribe Anacardieae, same with the genus Mangifera. In this study, it is the first report of the genetic diversity assessment of lacquer species of the genus Gluta. The result showsG. lacciferathat is a sister group of G. glabra to be consistent with its flower morphological characteristics having closely by hairy ovary stalk [32].By the past, G. glabracouldprovided sap to use a material for lacquer ware handicraft, which was mixed with other sap of lacquer species including G. laccifera, its sap was called in “RakChaiya”, name of its origin from Chaiya district, Surat Thani province where locate in peninsular Thailand. Its sap was super quality, whereof the artisan needed to use a material for lacquer ware making. Both G. lacciferaand G. glabrain under name of Burmese lacquer or ‘Rakyai’ (G. usitata) by Thai craftsmen, whereas three Glutaspecies are closely morphological characters with having 30 stamens, fruit having 5-6 large wings while other Glutaspecies absent wing[13].  The natural gene pool Gluta species is poorly represented in the natural habitats. The area of adaptation of Gluta species is found often forest fragments which are highly vulnerable to deforestation and urbanization. Thus, conservation of the Gluta species gene pool is absolutely essential to revive and maintain genetic variability in natural conditions.

4.                  Conflict of Interest

None.

5.                  Conclusion

This study on genetic diversity of 71 selected Gluta lacquer clones from northeastern Thailand analyzed using by SCoT markers. Ten suitable of 45 SCoT primers showed clearly polymorphisms in ranges 400 - 3,000 bp, via; SCoT38, SCoT43, SCoT47, SCoT48, SCoT54,SCoT55, SCoT58, SCoT61, SCoT63 and SCoT67 primers. The result showed total 57 bands, average 5.7 bands per primer and 37 polymorphic bands or 69%. Jaccard’s similarity coefficient was ranged from 0.36 - 0.97.  A dendrogram constructed using unweighted pair group method with arithmetic mean (UPGMA) of Gluta lacquer clones classified into 5 clusters at 0.61 of genetic similarity coefficient.

6.                  Acknowledgements

The work is a part of the project ‘Study on productivity and sustainability of lacquer sap for the revitalization of Thai wisdom Code P-T (D): 22.55. Supported by Kasetsart University Research and Development Institute (KURDI).

Figure 1: Application products generated by SCoT 63 with13 clones of Gluta species. Lane M shows the 100 bp DNA ladder plus and Lane 1-13; A21, A25, A24, A39, A22, A23, A42, A26, A27, A28, A29, A30, A31, respectively.

Figure 2:  Dendrogram of the 71 Gluta samples studied based on UPGMA analysis of 10 SCoT primers using the Jacquard’s similarity coefficient.

Table 1: List of the Glutasamples in this study.

Table 2: List and sequences of each primer and their total bands number, polymorphic band number, polymorphic band percentage of 71 samples of Glutasamples as revealed by SCoT markers.

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