Breeding Science 60: 412–418 (2010) doi:10.1270/jsbbs.60.412 Assessment of genetic diversity in cassumunar ginger (Zingiber cassumunar Roxb.) in Thailand using AFLP markers Maytinee Kladmook1), Sopida Chidchenchey1) and Vichien Keeratinijakal*2) 1) National Center for Agricultural Biotechnology, Kasetsart University, Bangkok 10900, Thailand 2) Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand The genetic relationship among cassumunar gingers (Zingiber cassumunar) in Thailand was assessed by am- plified fragment length polymorphism (AFLP). Twelve AFLP primer combinations generated a total of 309 fragments, of which, 242 bands were polymorphic with an average of 20.2 bands per primer pair. Genetic similarities were obtained using Jaccard similarity coefficients, and a phylogenetic tree was constructed using the UPGMA clustering method. Pairwise similarity estimated between cassumunar gingers ranged from 0.7644 to 1.00 with an average of 0.879. Cluster analysis divided the samples into five groups with a high co-phenetic correlation value (r = 0.99). Genetic variability within and among collection regions was estimat- ed by analysis of molecular variance (AMOVA). High molecular variance (84%) was found within samples from the same region. The results implied dispersal of plant materials between collection regions. The genetic similarity assessed by AFLP showed that there are duplicate accessions in the germplasm collection. This ge- netic information is very useful for germplasm maintenance and a crop improvement program. Key Words: AFLP markers, genetic diversity, Zingiber, Z. cassumunar. Introduction tion of Z. cassumunar collections is not easy, because some morphological traits are affected by the environmental con- Zingiber cassumunar Roxb., locally called “Phlai” in Thai- ditions under which the plants are grown. Conversely, the land, is an important medicinal plant in Southeast Asia. The utilization of molecular markers has been effective in evalu- essential oil from the rhizome is used for reducing inflam- ating genetic diversity independent of environmental influ- mation from injuries, sprains in muscles and joint issues. ences and the stage of plant growth. The results from pharmacological study show that the essen- Nowadays, molecular markers are powerful tools for the tial oil has several properties, such as an antiseptic, antitoxic evaluation of genetic diversity and the easy identification of and strong anti-inflammatory effect (Kuroyanagi et al. 1980, species. Genetic diversity of the Zingiberaceae has been Jeenapongsa et al. 2003, Jitoe et al. 1994, Masuda and Jitoe studied also using DNA markers. Many researchers have fo- 1994, Panthong et al. 1990, 1997, Tuntiwachwuttikul et al. cused on species identification or assessment of the genetic 1980, 1981). With the rising popularity of herbal products as relationship among species of the genus Zingiber. Kress et drugs and cosmetic being reported (Akerele 1993, Cupp al. (2002) used molecular sequence data to generate hypoth- 1999, Riewpaiboon 2006), Z. cassumunar has high potential eses on the phylogenetic relationships among the genera of to become a new commercially valuable plant. Zingiberaceae. Theerakulpisut et al. (2005) classified mem- The herbal product chain begins with the selection of cul- bers of the genus Zingiber by RAPD markers. The genetic tivars. The properties of the plant should be identified and diversity information within Z. cassumunar has been rarely measured against the target profile of the final product to reported. guarantee that the quality of the product is not compromised. The amplified fragment length polymorphism (AFLP) Plant breeding can help to create different varieties of a spe- technique, developed by Vos et al. (1995), is an effective, cies that have more suitable characteristics, both in terms of cost efficient and reproducible method for revealing DNA chemical profile and agronomical characteristics, and these polymorphisms. The current study used AFLP markers to varieties can increase yield and reduce cost. Germplasm col- elucidate the phylogenetic relationships among 132 acces- lection and diversity analysis of Z. cassumunar are prerequi- sions of Z. cassumunar and its related species. Because sites for a breeding program. There is insufficient data on the cassumunar ginger is vegetatively propagated, there might identification of varieties; the morphological characteriza- be duplicate samples in the germplasm collection. Therefore, another aim of this research was to investigate whether it Communicated by K. Okuno was possible to eliminate duplication in the accessions using Received May 10, 2010. Accepted October 18, 2010. the AFLP marker. *Corresponding author (e-mail: [email protected]) Genetic diversity of cassumunar gingers in Thailand 413 Materials and Methods Table 1. Average number of bands, number of alleles and proportion of polymorphic bands obtained for the 132 accessions from 12 selec- Plant material and genomic DNA extraction tive primer combinations A total of 132 accessions, including three samples of Total band Polymorphic % polymorphic Primer combination Z. zerumbet and a Zingiber sp., locally called “Phlai Pah” No. band No. band (which means “wild cassumunar ginger”), as outgroups, dis- E-AAC/M-CTG 27 20 74.07 tributed throughout Thailand were collected. The location of E-ACC/M-CAC 31 24 77.42 collection areas followed the floristic regions and provinces E-ACC/M-CTA 26 18 69.23 of Thailand (Fig. 1). The cassumunar accession numbers and E-ACC/M-CTC 22 14 63.64 collection sites are shown in Supplemental Table 1. The E-ACC/M-CTT 28 20 71.43 morphological characterization was conducted at the E-ACG/M-CAC 30 25 83.33 National Corn and Sorghum Research Center, Pakchong, E-ACG/M-CAT 26 20 76.92 Nakhon-Ratchasima province. The following characteristics E-ACG/M-CTC 24 23 95.83 in each accession were measured: plant height, number of E-AGC/M-CTG 25 24 96.00 tillers per clump, leaf width and length, inflorescence stalk E-AGC/M-CTT 26 19 73.08 E-AGG/M-CAC 17 11 64.71 length and inflorescence length/width ratio. E-AGG/M-CTG 27 24 88.89 Total genomic DNA was extracted from leaf tissue ac- Total 309 242 Average 25.75 20.16 77.88 E = pre-amplification primer (GACTGCGTACCAATTC) of EcoRI; M = pre-amplification primer (GATGAGTCCTGAGTAA) of MseI. cording to the CTAB method, following the procedures of Doyle and Doyle (1990). The concentration of DNA was quantified by measuring the absorbance of UV light (260 nm) by spectrophotometer and then adjusting the con- centration to 50 ng/μL for AFLP analysis. AFLP analysis Genomic DNA (0.25 μg) was digested with 2.5 units of EcoRI and MseI (Biolabs, Australia) in a final volume of 25 μL containing digestion reaction solution (50 mM potas- sium acetate, 20 mM Tris-acetate pH 7.9, 10 mM magne- sium acetate, 1 mM dithiothreitol, 0.1 mg/mL BSA). After mixing, the DNA samples were incubated for 3 h at 37°C. Ligation of EcoRI and MseI adaptors was performed by mixing 25 μL of double digested DNA and 25 μL of ligation solution (1unit of T4 DNA ligase, 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP). The mix- ture was then incubated at 25°C for 2 h. The pre-selective amplification reaction was performed using 2 μL of digestion/ligation reactions, in 25 μL of PCR reaction containing 200 mM Tris-HCl pH 8.4, 500 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 pmol of EcoRI and MseI adapter-directed primers (each possessing a single selective base, E + 1; M + 1) and 1 U of Taq DNA poly- merase (Invitrogen, Brazil). PCR reactions were performed with the following profile: 94°C for 3 min, 30 cycles of 30 s denaturing at 94°C, 30 s annealing at 56°C and 60 s exten- sion at 72°C, ending with 5 min at 72°C to complete exten- sion. After checking for the presence of a smear of frag- Fig. 1. Map showing geographical origin of accessions of ments (100–1000 bp in length) by agarose electrophoresis, × Z. cassumunar, Phlai Pah and Z. zerumbet. NE = North-East; the amplification product was diluted 20 times in 0.1 TE. C = Central; N = North; E = East; PEN = Peninsular Thailand; Selective amplification (second PCR) of the diluted pre- SW = South-West and SE = South-East. The numbers indicate the total amplification products was carried out using 12 primer number of samples collected in each province. combinations (Table 1). Selective PCR reactions were 414 Kladmook, Chidchenchey and Keeratinijakal performed with the following profile: 94°C for 60 s, 36 cycles fit between the dendrogram clusters and the similarity ma- of 30 s denaturing at 94°C, 30 s annealing and 60 s extension trix from which they were derived. at 72°C, ending with 10 min at 72°C to complete extension. The dendrogram based on the UPGMA method consisted Annealing was initiated at a temperature of 65°C, which was of two major clusters (Fig. 2). Cluster I comprised cassumunar then reduced by 0.7°C for the next 12 cycles and maintained ginger samples and cluster II included the Phlai Pah (acces- at 56°C for the subsequent 23 cycles. The second PCR prod- sion ‘w’) and three Z. zerumbet samples (accession nos. 52, ucts were mixed with 10 μL of loading dye (98% form- 103 and 124) which were used as outgroups. Cluster I could amide, 10 mM EDTA, 0.01% w/v bromophenol blue and be divided into five subgroups (subgroups Ia, Ib, Ic, Id and 0.01% w/v xylene cyanol), denatured at 95°C for 5 min and Ie) at a cut-off genetic similarity value of about 0.88. The separated on 6% denaturing polyacrylamide gels (6% poly- typical AFLP patterns of each cassumunar ginger subgroup, acrylamide 29 : 1, 7 M urea) in 1× TBE buffer.