Agr. Nat. Resour. 55 (2021) 105–112 AGRICULTURE AND NATURAL RESOURCES Journal homepage: http://anres.kasetsart.org Research article Genetic diversity of ‘Bangle’ (Zingiber montanum (J.Koenig) Link ex A.Dietr.) inferred from sequence-related amplified polymorphism markers Marlina Ardiyania,†,*, Seni Kurnia Senjayaa,†, Anshari Maruzyb, Yuli Widiyastutib, Lulut Dwi Sulistyaningsiha, Susilaa a Botany Division, Research Center for Biology, Indonesian Institute of Sciences, Bogor 16911, Indonesia. b Medicinal Plant and Traditional Medicine Research and Development Centre, National Institute of Health Research and Development, Karanganyar 57792, Indonesia. Article Info Abstract Article history: Zingiber montanum (J.Koenig) Link ex A.Dietr. known in Indonesia as ‘Bangle’ is widely Received 29 February 2020 cultivated there in addition to other Zingiber species, such as Z. officinale Roscoe and Z. zerumbet Revised 27 July 2020 Accepted 28 October 2020 (L.) Sm. Despite the rich properties of its rhizomes as an anti-oxidant and anti-inflammatory, Available online 26 February 2021 knowledge of its genetic diversity is still scarce. Understanding genetic diversity is important for conservation purposes and to improve the genetic contents. Consequently, genetic diversity of Z. Keywords: montanum was analyzed using sequence-related amplified polymorphism (SRAP) markers. In DNA profiling, total, 54 Z. montanum samples collected from 12 regions in four provinces were analyzed using Genetic diversity, Indonesia, eight SRAP primer pairs which amplified the open reading frames region. The results showed that Zingiberaceae Z. montanum had high genetic diversity with mean (± SD) values for Nei’s genetic diversity and the Shannon information index of 0.257 ± 0.172 and 0.400 ± 0.223. Even though Z. montanum is a clonal species, it was able to maintain high genetic diversity. The analysis of molecular variance showed that the genetic variation occurred mostly within the population. High genetic diversity and low differentiation among populations could suggest that rare sexual reproduction has played an important role in Z. montanum population history. Bayesian model-based clustering was congruent with the unweighted pair-group method of averages dendrogram at the sub-cluster level. The Z. montanum populations were grouped into three clusters with strong correlation within geographic locations. Based on the results, germplasm should be collected and maintained for at least one individual from each of the three clusters. Introduction probably native to India and Southeast Asia and is commonly grown in villages (Theilade, 1996). Z. montanum is a diploid with 2n = 2x = 22 Zingiber montanum (J.Koenig) Link ex A.Dietr. (family Zingiberaceae) (Raghavan and Venkatasubban, 1943; Larsen et al., 1998) presenting known as ‘Bangle’ in the Javanese language is a medicinal plant its inflorescence on a radical, erect peduncle and having spherical widely used in Southeast Asia (Wolff et al., 1999). Z. montanum is pollen (Valeton, 1918). The flowers are tubular and contain nectar and like in other Zingiberaceae species last only for 1 d (Larsen et al., † Equal contribution. * Corresponding author. 1998). The plant is the same as most other Zingiberaceae, flowering E-mail address: [email protected] (M. Ardiyani) online 2452-316X print 2468-1458/Copyright © 2021. This is an open access article, production and hosting by Kasetsart University of Research and Development Institute on behalf of Kasetsart University. https://doi.org/10.34044/j.anres.2021.55.1.14 106 M. Ardiyani et al. / Agr. Nat. Resour. 55 (2021) 105–112 between July and September and fruiting from November to January Z. montanum is a medicinal plant that will continue to be exploited. (Theilade, 1996). There is no detailed information about Z. montanum An evaluation of its genetic diversity is very important. This study pollinators but its relative species in Kalimantan are categorized into analyzed the genetic diversity of Z. montanum from four provinces three groups based on their pollinator guilds, namely spiderhunters, in Indonesia using sequence-related amplified polymorphism (SRAP) Amegilla bees and halictid bees (Sakai and Kato, 1999). markers (Li and Quiros, 2001). SRAP belongs to the targeted In Indonesia, Bangle is widely cultivated as a minor crop planted fingerprinting marker group and is a multilocus marker that is in backyards and has been reported to be cultivated in Java, Moluccas, produced semi-randomly and targets various sequence regions in the Sibolangit (Sumatra) and Malacca since the colonial era (Valeton, genome (Poczai et al., 2013). A SRAP marker was developed to recognize 1918). The medicinal properties of Bangle are known by the Sundanese the exon, intron, spacer and promoter region (Li and Quiros, 2001). to cure fever, postpartum drugs, dermatitis, hookworm, diarrhea and Knowledge of genetic diversity of Bangle is expected to be useful as gastritis (Roosita et al., 2008). The Dayak Kenyah people have used a foundation for the breeding and development of Bangle in Indonesia. Bangle for a wide range of treatments, including gynecological, gastrointestinal, respiratory and muscular problems (Ellen and Puri, Materials and Methods 2016). Recent study reported that the essential oil of Bangle is a potential source of (E)-1-(3’,4’-dimethoxyphenyl) buta-1,3-diene, Samples terpinen-4-ol and sabinene for pharmaceutical products (Verma et al., 2018). In addition, Bangle is a component of Indonesian herbal In total, 54 samples of Z. montanum were used in this study to medicine (‘Jamu’), which has been industrialized. investigate the genetic variation and population structure. Samples The National Institute of Health Research and Development were collected from 12 locations in four provinces in Indonesia (NIHRD) has inventoried Indonesian medicinal plants including (Table 1) during RISTOJA exploration conducted by the Medicinal Z. montanum through the Riset Tanaman Obat dan Jamu (RISTOJA) Plant and Traditional Medicine Research and Development Centre, or Research on Medicinal Plants and Jamu project and collected NIHRD from 2015 to 2017. The samples were obtained from limited medicinal plants from all over Indonesia. Development of medicinal provinces since only few ethnic communities used the plant. Medium- plant germplasm has been pioneered by the Medicinal Plant and aged, healthy leaves of approximately 2 cm × 2 cm were cut into small Traditional Medicine Research and Development Centre, NIHRD. To pieces, then stored in a tea bag before being placed in a container filled gain benefit from these germplasms, assessment and characterization with silica gel. of genetic diversity is very important for planning meaningful breeding strategies and medicinal plant improvement. DNA extraction and SRAP analysis Genetic diversity of Z. montanum in Indonesia is poorly known. Research on genetic diversity of Z. montanum has been carried out Total DNA was isolated from the silica-dried leaves following the on genotypes collected from various locations in Thailand using 29 GeneJet Plant Genomic DNA Purification Mini Kit (ThermoFisher RAPD primers and showed high molecular variance (87%) within its Scientific) protocol with slight modification by adding proteinase K to accession (Bua-in and Paisooksantivatana, 2010). The same result was the sample in the second step of isolation to increase the concentration observed by Kladmook et al. (2010) who assessed genetic diversity of DNA yields. The isolated DNA was diluted with nuclease-free of Z. montanum from Thailand using 12 amplified fragment length water to a final concentration of 10 ng/µL. Then, 16 SRAP primer polymorphism (AFLP) primers and found high molecular variance combinations were screened of primer pairs of four forward primers (84%) within samples from the same region. (Me1, Me2, Me3, and Me4) (Li and Quiros, 2001) and four reverse Table 1 Genetic diversity of 12 Zingiber montanum populations Population Locality Sample size He I PPL GST Nm Jawan West Kalimantan 5 0.127 ± 0.191 0.187 ± 0.275 33.58% - - Kayanath West Kalimantan 5 0.084 ± 0.158 0.126 ± 0.234 23.36% - - Kore West Nusa Tenggara 3 0.096 ± 0.167 0.146 ± 0.243 29.20% - - Pattinjo South Sulawesi 5 0.225 ± 0.207 0.334 ± 0.292 60.58% - - Bonerate South Sulawesi 5 0.147 ± 0.198 0.218 ± 0.285 40.15% - - Padoe South Sulawesi 5 0.223 ± 0.201 0.334 ± 0.282 63.50% - - Kalatoa South Sulawesi 5 0.137 ± 0.190 0.205 ± 0.275 38.69% - - Rongkong South Sulawesi 3 0.071 ± 0.152 0.105 ± 0.223 18.98% - - Tialo Central Sulawesi 5 0.137 ± 0.200 0.201 ± 0.286 35.04% - - Lauje Central Sulawesi 5 0.083 ± 0.168 0.124 ± 0.237 23.36% - - Bungku Central Sulawesi 5 0.097 ± 0.161 0.150 ± 0.238 31.39% - - Ledo Central Sulawesi 3 0.072 ± 0.161 0.104 ± 0.231 17.52% - - Species Level 54 0.257 ± 0.172 0.400 ± 0.223 98.54% 0.5112 0.4781 He = Nei’s gene diversity; I = Shannon’s information index; PPL = percentage of polymorphic loci; GST = population differentiation value; Nm = estimated gene flow Values are shown as mean ± SE M. Ardiyani et al. / Agr. Nat. Resour. 55 (2021) 105–112 107 primers (Em1, Em2, Em3, and Em4) (Li and Quiros, 2001). Primer admixture model, the correlated allele frequencies model and no prior pairs which produced polymorphic bands (Table 2) were used for population information. The optimum number of K was calculated further analysis. The polymerase chain reaction (PCR) was performed using the ΔK method of Evanno et al. (2005). Subsequently, each in 15 µL of reaction mixture containing 10 ng DNA template, 7.5 µL individual/sample was assigned to each subpopulation by running DreamTaq Green PCR Master Mix (2x) (ThermoFisher Scientific) and STRUCTURE with the optimum K value, burn-in length of 10,000 2 µM of both forward primer and reverse primer. The PCR program and run length of 100,000 MCMC replications. Principal component for amplification was set following Li and Quiros (2001) with pre analysis (PCA) using Jaccard’s similarity coefficient was performed to denaturation at 95°C for 5 min, five cycles of denaturation at 95°C for confirm similarity among the 54 samples.