MATERIALS AND METHODS

Plants materials: Young leaves of 15 individuals of N. nucifera were randomly collected from 5 populations in various geographical areas within the provinces of Udon Thani, Sakon Nakhon, Khon Kaen and Loei, all 4 of which are provinces in Northeastern Thailand (NE) and within Suphan Buri province, Central Thailand (C) (Table 1). Three individuals were selected in each province which are over 50-60 km apart distances. The distances of Khon Kaen to Udon Thani, Udon Thani to Sakon Nakhon, Sakon Nakhon to Khon Kaen and Khon Kaen to Suphan Buri are 120, 160, 200 and 560 km, respectively. The province locations are shown on a map of Thailand in Fig. 1. Leaf materials were collected and immediately dried using silica gel and then stored at -70°C until the DNA was extracted. Out group for this study is Nymphaea lotus L. Sample collections were done in 2005 and DNA extraction, ISSR analysis and ISSR data analysis were conducted in 2006 at Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.

DNA extraction: Genomic DNA was extracted from dried leaves using the Qiagen DNeasy mini-kit. The DNA was checked by subjecting it to 0.8% agarose gel electrophoresis stained with ethidium bromide. The quality and quantity of the DNA samples were determined by a gel documenting instrument. DNA samples extracted from three individuals from each province were pooled into a study sample, thus 5 pooled DNA samples were used for further analysis. Then, DNA samples were diluted in TE to a final concentration of 20 ng μL^-1. These dilutions were used as DNA templates in PCR reactions.

Table 1:	Studied samples, collection sites and morphological variations of Nelumbo nucifera Gaertn

Fig. 1:	A map of Thailand indicating sample collection provinces (•) of nu1 (Udon Thani), nu2 (Sakon Nakhon), nu3 (Khon Kaen), nu4 (Loei) in northeastern (NE) Thailand and nu5 (Suphan Buri) in central (C) Thailand

Table 2:	Summary of ISSR primers, number of bands scored, number of polymorphic bands and percentage of polymorphisms for amplification profile of five individuals of Nelumbo nucifera Gaertn

ISSR analysis: Amplification reactions were performed using a PCR machine (Gene Amp PCR system 9700, Applied Biosystems). Amplifications were carried out in 25 μL reactions consisting of Taq reaction buffer, 2 mM of MgCl₂, 0.3 mM of each dNTP, 0.3 μM primer, 1.25 units of Taq DNA polymerase (Invitrogen) and 5 ng of DNA template. Thirty-five ISSR primers were used in PCR reactions. The 16 ISSR primers that successfully amplified clear bands for N. nucifera are shown in Table 2.

The reaction mixture was incubated at 94°C for 3 min and the amplification was performed with the following thermal cycles: 35 cycles of denaturation for 1 min at 94°C, 2 min annealing temperature Tm-5, 2 min at 72°C and 7 min final extension at 72°C. Amplification products were detected by agarose gel electrophoresis in TAE buffer (0.4 M Tris, 0.114% acetic acid 1 mM EDTA pH 8.0) and visualized by ethidium bromide staining.

ISSR data analysis: The total number of ISSR bands discerned from the agarose gel was documented as diallelic characters: present = 1, absent = 0; as the ISSR markers are considered the dominant markers. The resulting bands were used to construct a dendrogram following the UPGMA with the Fingerprinting II program version 3.0 (Bio Rad). As noted earlier the outgroup species is Nymphaea lotus. The similarity values are obtained from the dendrogram. The percentage of polymorphic loci was calculated with the Shannon’s diversity index (H) for each population, H = -Σ pi ln pi, where, pi is the frequency of a given ISSR band. Genetic evenness (E) is calculated from genetic diversity/ln species richness (ln 5 = 1.61) (Nei, 1973, 1978). Mantel tests (r) (Mantel, 1976) were performed to analyze the effects of geographical distance on genetic distance by using the formula:

where:

RESULTS

Thirty-five primers were screened and 16 informative ISSR primers produced a total of 141 bands with sizes ranging from 200 to 2000 bp and an average of 8.81 bands per primer. Of these bands, 46.09% (65 bands) were polymorphic. Percentages of Polymorphic Bands (PPB) for each primer ranged from 0 to 100%. Primer (GT)₆GG generated the highest number of bands (20 bands). The minimum number of bands (5) was produced by primer (CA)₉A (Table 2). Figure 2 shows the banding pattern using the primer (AC)₈G, one of the 16 informative primers which demonstrates the differences of banding pattern between individuals and geographical areas in a population of N. nucifera.

The dendrogram (Fig. 3) clearly distinguishes the studied samples into two groups, one including nu1, nu2 and nu3 and the second comprising nu4 and nu5 without consideration of the flower color and the absence or presence of petaloid staminodes and petaloid stamens. The similarity (S) values of a species N. nucifera (nu1-nu5) range display from 84.10 to 95.00%. The S values support the studied samples to be in three groups. The first group, nu1-nu2, without petaloid staminodes and petaloid stamens, always grows in natural conditions, having white and pink tepals and has the highest S value of 95%. The second group, nu3-nu4, with S value of 88.90%, are with petaloid staminodes and petaloid stamens. They are popularly accepted for commercial growing for religious and ornamental purposes because of producing many flowers and having white tepals. The third group comprising populations, nu3-nu5, with petaloid staminodes and petaloid stamens, is also popularly accepted for commercial growing because of high flower productivity and used for religious and ornamental activities, having white and pink tepals and a high S of 84.1% (Table 3).

Table 3:	Genetic similarity values of Nelumbo nucifera Gaertn. population

Table 4:	Genetic diversity (H) and Evenness (E) values of Nelumbo nucifera Gaertn. population

Fig. 2:	ISSR profile of Nelumbo nucifera Gaertn. and Nymphaea lotus with primer (AC)8G

The genetic diversity of the population species of the five studied samples collected in different provinces is 1.58 (Table 4). The value reflects the broad range of distribution sites of the species. Genetic richness is 5, ln genetic richness (ln 5) is 1.69, so the genetic evenness is 0.98 (Table 4). This value supports that all studied samples have nearly equally genetic abundance.

The Mantel test shows a significant positive correlation between geographical and genetic distances among all five studied samples, r = 0.061. This reveals that geographical distance is an important factor affecting the genetic distance between populations.

Fig. 3:	A dendrogram constructing from 16 ISSR primers determined by UPGMA (Fingerprinting II, Bio Rad) to clarify the genetic relationships of Nelumbo nucifera Gaertn. Nymphaea lotus is an out group

DISCUSSION

The highest S value of 95% in natural variety, nu1 and nu2, indicates a very good genetic interchange or gene flow interchange between populations, without human factor disturbance. In contrast, S value of lower than 95% is due to the interference from having human activities for making optimal conditions to produce many more flowers for use in religious and ornamental activities. However, the S of N. nucifera is in a range of a species, 84.1-95% following Weier et al. (1982) and Rost et al. (1984).

The genetic diversity using ISSR markers indicates a high polymorphic value of 46.09 % in the geographically separated samples. The genetic diversity in a population is still high at 1.58. The great value of genetic diversity supports the broad range of distribution sites of N. nucifera, which would require high genetic diversity to adapt to the environmental variations that can be found between NE and C Thailand.

Actually, the number of individuals for genetic diversity evaluation should be 20-30 individuals and cover distant distribution areas. However, the individuals per sample and distributed collection areas of these studied samples indicate the genetic diversity in NE and C Thailand. This shows that the genetic diversity of N. nucifera should be able to fit environmental variation (Fu et al., 2003). High genetic diversity is important, allowing species to adjust to their ever-changing environment, whether these changes are due to the natural or human factors (Chamberlain and Hubert, 2001).

The genetic evenness value, 0.98, supports that all studied samples have nearly equally genetic abundance. The Mantel test value of 0.06 confirms the significant positive correlation among all 5 studied samples. In conclusion, the geographical distances between samples affect the genetic distances.

The method of taking extensive genetic diversity and similarity by collecting 3 individuals into a studied sample is an alternative method for saving chemicals and working time as showed by good values of genetic diversity and similarity.

While the study sample is small and dose not cover all parts of Thailand, a broad genetic diversity is represented because the collection sites cover a wide range of areas from NE to C Thailand and a number of individuals. Accordingly, the resulting values agree with conclusions found in the cited studies. These factors suggest that the results of this study may be considered valid.