(Zingiber Cassumunar Roxb.) in Thailand Using AFLP Markers

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 amplified 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 estimated 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 genetic 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 concentration 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 extension at 72°C, ending with 5 min at 72°C to complete extension. 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. The gels wild cassumunar ginger and Z. zerumbet are shown in Fig. 3. were pre-run at 300 V for about 30 min before 10 μL of the The clustering of the accessions based on genetic similarity mix was loaded. Gels were run at 300 V for about 2.5 h. The did not correlate with their region of origin. AFLP fragments were visualized by silver staining (Benbuasa The distribution of genetic diversity within and between et al. 2006). regions was explored using AMOVA (Table 2). Although AMOVA displayed significant divergence among the col- Data analysis lection regions (Fst = 0.158, P-value = 0.010), most of the For the diversity analysis, each PCR product was as- genetic variation was found within collection regions (84%). sumed to represent a single locus and only polymorphic The mean values of the morphological characters used in bands were scored as present (1) or absent (0). A binary ma- the characterization of each subgroup are shown in Table 3. trix was imported into NTSYS-pc version 2.20k (Rohlf There was a significant difference (p ≤ 0.05) between the 2005) for cluster analysis. Genetic similarity among all ac- cassumunar ginger subgroups for four measured characters: cessions was calculated according to Jaccard’s Similarity In- leaf width, leaf length, inflorescence stalk length and inflores- dex (JSI) (Jaccard 1908) by the SIMQUAL subprogram, and cence length/width ratio. The inflorescence pictures of each the SAHN subprogram was used for cluster analysis by the cassumunar ginger subgroup, Phlai Pah and Z. zerumbet are UPGMA method (unweighted pair-group method with arith- shown in Fig. 4. metic means) (Sneath and Sokal 1973). A co-phenetic ma- The genetic similarity between cassumunar gingers as- trix was produced using the hierarchical cluster system, by sessed by AFLP markers revealed that there were several means of the COPH routine, and correlated with the original duplicate accessions in the germplasm collection. The num- distance matrices for the AFLP data, in order to test for ber of accessions could be reduced from 128 to 73. Of all 55 agreement between the cluster in the dendrogram and the JSI duplicate accessions, 27 accessions were collected from the matrix. The genetic relationships between the Zingiber ac- same or neighboring districts. cessions, based on the results from cluster analysis, were portrayed in the form of dendrogram. The polymorphic in- Discussion formation content (PIC) was calculated by applying the sim- plified formula of the expected heterozygosity. The analysis The genetic diversity of Z. cassumunar germplasm was eval- of molecular variance (AMOVA) was calculated by the uated by AFLP markers. A high level of genetic similarity of computer program GenALEx 6 (Peakall and Smouse 2006). cassumunar gingers was found. The same result was reported by Bua-in and Paisooksantivatana (2009), who used Results RAPD markers to estimate the genetic diversity of 32 cassumunar ginger samples from Thailand. This result is A total of 242 polymorphic bands (78.32% of the total am- also comparable with other clonally propagated species. plified bands), ranging from 100 to 1,100 bp was scored. Wahyuni et al. (2003) reported the average pair-wise gene- The average number of polymorphic bands per primer was tic similarity of ginger (Z. officinale Rosc.) revealed by 20.2, while the range for twelve primers was 11 to 25 AFLP was 0.801. Li et al. (2006) reported the clonal plant, (Table 1). On inclusion of Phlai Pah and some outlier spe- Eichhornia crassipes, in China had low genetic variation. cies, the result obtained by Jaccard’s coefficient showed that The co-phenetic correlation coefficient (r-value) between the genetic similarity varied from 0.2457 to 1.00. The lowest the AFLP-based data dendrogram and the similarity matrix genetic similarity among accessions of Z. cassumunar was clustering was 0.99, demonstrating a good fit between the 0.7644 (between accessions 4 and 119). The mean similarity dendrogram clusters and the similarity matrix from which was 0.879. The PIC value ranged from 0.015 to 0.500 (mean they were derived. The clustering of the accessions based on 0.115). UPGMA analysis of genetic similarity estimates was genetic similarity did not correlate with their region of ori- performed using NTSYS version 2.20k and the results are gin. This result might be caused by the distribution of plant shown in Fig. 2. The co-phenetic correlation coefficient (r- materials between collection regions. However, all of the ac- value) between the AFLP-based data dendrogram and the cessions from peninsular Thailand were clustered together. similarity matrix clustering was 0.99, demonstrating a good This result indicated that there was no plant material transfer Genetic diversity of cassumunar gingers in Thailand 415

Fig. 2. UPGMA phylogenetic tree of 132 accessions by AFLP analysis using Jaccard’s similarity matrices. between cassumunar gingers from the peninsular to other re- The dendrogram based on the UPGMA method consisted gions. These results are relevant to the study of cassumunar of two major clusters (Fig. 2). Cluster I comprised all ginger samples using RAPD markers, which reported that 9 cassumunar ginger samples, which could be divided into five of 11 Z. cassumunar accessions from the peninsular were subgroups. Interestingly, these groupings obtained within grouped in the same cluster (Bua-in and Paisooksantivatana cluster I are relevant to some morphological differences of 2009). the genotype. Most of the samples in subgroup Ia were late 416 Kladmook, Chidchenchey and Keeratinijakal

most all samples had bracts tipped green, while the others had a purple tip (Fig. 4D). The inflorescence length/width ratio of subgroup Id was significantly higher than other subgroups. The mean values of the morphological characters used in the characterization of each subgroup are shown in Table 2. There was a significant difference (p ≤ 0.05) between the subgroups for four measured characters: leaf width, leaf length, inflorescence stalk length and inflorescence length/width ratio. This confirmed that the accessions in each subgroup were morphologically different. The Zingiber sp. sample used in this study was locally called “Phlai Pah”, which means “wild cassumunar ginger” since its habitat is in the forest. As shown in Fig. 3, the AFLP pattern of Phlai Pah differed from that of cassumunar gingers. The result from the dendrogram revealed that Phlai Pah was grouped in cluster II, which included three Z. zerumbet samples. The higher genetic similarity between Phlai Pah and cultivated cassumunar gingers was expected. Further- more, a high genetic distance between Z. zerumbet and Phlai Pah (genetic distance = 0.64) was observed. The morphological characteristics of Phlai Pah, such as plant height and inflorescence characters, were also significantly different from cultivated cassumunar gingers and Z. zerumbet, this Phlai Pah may have been misunderstood as a wild relative of cassumunar ginger. Presently, there are no taxonomic data of this plant. Future study on taxonomic identification of this plant is needed. The wild relatives of cultivated cassumunar ginger may provide valuable genetic resource for a breeding program. The potential of Z. zerumbet as a soft-rot disease resistance donor for genetic improvement of ginger has been Fig. 3. The AFLP pattern of each cassumunar ginger subgroup, Phlai reported (Kavitha and Thomas 2007, 2008). Pah and Z. zerumbet; (A) subgroup Ia, (B) subgroup Ib, (C) subgroup The distribution of genetic diversity within and between re- Ic, (D) subgroup Id, (E) subgroup Ie. (F) Phlai Pah and (G) Z. zerumbet gions was explored using AMOVA (Table 3). High molecu- using the E-ACC/MCAC primers. The numbers labeled in each lane lar variance (84%) within collection regions of Z. cassumunar indicate the accession numbers of samples (M = 100 bp DNA stan- dard). accessions was revealed and this result also showed the significant divergence among the samples from the six collection regions. This indicated dispersal of plant material from flowering types and the inflorescence shapes were ellipsoid other regions. A similar result was reported by Jatoi et al. with a long, accumulated apex (Fig. 4A). The average height (2008) and Bua-in and Paisooksantivatana (2009). of the pseudo-stem and the size of the leaf blade in subgroup The redundant cost of field maintenance caused by dupli- Ib accessions was higher than others, the average width of cate accessions is an important problem in large collections inflorescence was slightly greater than in subgroup Ia of germplasm. The genetic similarity between cassumunar (Fig. 4B). All of the white-stripe leaf samples, which locally gingers assessed by AFLP marker revealed that there are are called “Phlai Khrueng”, and accessions with a yellow- several duplicate accessions in the germplasm collection stripe leaf were clustered in subgroup Ic. The average height studied. The number of accessions can be reduced from 128 of the pseudostem in subgroup Id was obviously low and al- to 73. The duplicate accessions detected by AFLP marker

Table 2. Mean of six morphological characters for five cassumunar ginger subgroups Pseudostem No. of tillers Inflorescence Inflorescence subgroup Leaf width Leaf length height per clump stalk length length/width ratio Ia 179.32 27.81 4.09 31.04 19.17 3.67 Ib 191.43 31.92 5.39 36.07 23.41 3.64 Ic 184.36 28.21 4.83 33.88 19.05 3.45 Id 165.50 32.10 3.83 29.29 21.00 3.94 Ie 180.00 29.00 4.70 31.00 21.00 3.74 Genetic diversity of cassumunar gingers in Thailand 417

Table 3. Analysis of molecular variance (AMOVA) to partition the variance within and among different collection sources Estimated % of molecular Region df SS MS F-value P-value variance variance Among collection regions 5 206.739 41.348 1.651 16% 0.158 0.010 Within collection regions 122 1070.636 8.776 8.776 84% Total 127 1277.375 10.427 100% df = degrees of freedom; SS = sum of squares; MS = mean sum of squares.

Fig. 4. The inflorescence of each cassumunar ginger subgroup, Phlai Pah and Z. zerumbet; (A) subgroup Ia, (B) subgroup Ib, (C) subgroup Ic, (D) subgroup Id and (E) subgroup Ie, (F) Phlai Pah (G) Z. zerumbet. were also identical in several key morphological characteris- redundancy in a germplasm collection. This genetic charac- tics. About half of the duplicate accessions were collected terization information is very useful for germplasm mainte- from the same or neighboring districts but the others were nance and for a crop improvement program. from remote sites. These findings also confirmed that plant materials were distributed between collection regions. In sev- Acknowledgements eral reports, the efficiency of DNA markers to identify and characterize the genetic diversity has been proven. Chiorato This work was supported by the National Research Council et al. (2006) used agromorphological and RAPD marker data of Thailand (NRCT) and National Center for Agricultural to detect unnecessary, duplicate, common bean accessions. Biotechnology (NCAB). The authors would like to thank Elameen et al. (2008) reported that the AFLP marker was Assistant Professor Dr. V. Hongtrakul for kindly providing able to detect duplicate accessions in sweet potato germ- laboratory facilities. The authors also thank Dr. Andrew J. plasm collections. Warner and Dr. P. Kongprakhon for review of the manu- The most common problem encountered by cassumunar script. ginger breeders is the identification of germplasms since the plant phenotypes are morphologically very similar. Future Literature Cited use of germplasms for breeding programs is limited if the collections are not properly identified. If the parental genotypes Akerele, O. (1993) Nature’s medicinal bounty: don’t throw it away. were identified and the genetic similarities of parental acces- World Health Forum 14: 390–395. sions were assessed, it would be easier for breeders to select Benbouza, H., J.M. Jacquemin, J.P. Baudoin and G. Mergeai (2006) Op- the parents. Thus, the results from this study provided useful timization of a reliable, fast, cheap and sensitive silver staining information for cassumunar ginger breeding in the future. method to detect SSR markers in polyacrylamide gels. Biotechnol. In conclusion, AFLP was used for a study of the genetic Agron. Soc. Environ. 10: 77–81. diversity in cassumunar gingers, as a first step towards its Bua-in,S. and Y.Paisooksantivatana (2009) Study of clonally propa- genetic improvement. The similarity indices, 0.7644 to 1.00, gated cassumunar ginger (Zingiber montanum (Koenig) Link ex Dietr.) and its relation of wild Zingiber species from Thailand revealed the close relationship among accessions in the germ- revealed by RAPD markers. Genet. Resour. Crop Evol. DOI: plasm collection. The dendrogram could divide the 128 sam- 10.1007/s10722-009-9479-2. ples into five subclusters, regardless of their region of origin. Chiorato, A.F., S.A.M. Carbonell, L.A.S. Dias, R.R. Moura, M.B. The diversity within each collection region was higher Chiavegato and C.A. Colombo (2006) Identification of common than between regions. The AFLP marker is an effective bean (Phaseolus vulgaris) duplicates using agromorphological and tool for genetic diversity assessment and the identification of molecular data. Genet. Mol. Biol. 1: 105–111. 418 Kladmook, Chidchenchey and Keeratinijakal

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