Diversity Analysis of 15 Hibiscus Accessions Based on RAPD Marker

Indonesia has many species of Hibiscus genus such as Hibiscus rosa-sinensis, kenaf, rosella, waru, sharon, and others. These species have similar flower morphology despite their different benefits. Flower morphology can be use as morphological marker characters to identify the genetic relationship in one genus of Hibiscus. However, morphological markers are less accurate because they are strongly influenced by the environment. Therefore, the molecular markers using plant DNA are considered to be more accurate than morphological characters. Random Amplified Polymorphic DNA (RAPD) analysis was applied to 15 Hibiscus genus. The result showed that 10 primers operon produced polymorphic bands. The high diversity was observed in control population i.e., H. cannabinus (K1), H. sabdariffa (K2), H. mutabilis (K3), H. syriacus (K4), H. schizopetalus (K5), while in H. rosa-sinensis, the diversity was low especially on the genotypes with the same color of flower. These results suggest that RAPD can be used to identify the diversity in plant population. The control species [H. cannabinus, H. sabdariffa, H. mutabilis, H. syriacus, and H. schizopetalus] showed high heterozygosity, suggesting high diversity were observed in these species. Based on principal coordinat analysis (PCA), two clusters were formed, the control species were clustered depend on life cycle of plant growing season either perennial or annual, while H. rosa-sinensis accessions were clustered based on the flowers color. The varian within population of H. rosa-sinensis accessions was higher than among population, suggesting that H. rosa-sinensis is cross pollinated plants due to the position of stigma is higher than anthers.


INTRODUCTION
Hibiscus belongs to the Malvaceae family.The Hibiscus genus is consist of 300 species with different growth characeristic ranging from small plants, shrubs, perennial crops, and annual crops spread over sub tropical and tropical area (Prasad, 2014).Hibiscus rosa-sinensis is able to grow and bloom throughout the year in both dry and rainy season, and even in a poor nutrient soil (Akpan, 2006cit Kuligowska et al., 2016).It has great variation of morphological characters such as flower colors and sizes, and the leaf shapes.The great variation in Hibiscus rosa-sinensis was observed because of crosses and mutation (Kadve et al., 2012).Another variation is observed in the flower shape either single and double petals (Hammad, 2009).Indonesia has many species of Hibiscus genus such as H. rosa-sinensis, kenaf, rosella, waru, sharon, etc.These species have similar flower morphology and different benefits such as traditional medicine (Pekamwar et al., 2013) or ornamental plant because of flower variation (Harvey, 2004).H. rosa-sinensis has been used for traditional medicine in India because its flower contents antioxidant, antifungal, anti-infectious, antimicrobial, anti-inflammatory, anti-diarrheic, and antipyretic compounds (Patel et al., 2012).Hibiscus consists of 300 species implying high diversity of this genera.Therefore, morphological and molecular marker can be used to reveal their phenotype and genotype diversities.
Flower morphology can be use as morphological marker characters to identify the genetic relationship in one genus of Hibiscus.However, morphological markers are less accurate because they are strongly influenced by the environment, requiring quite amount of time, and showing limited and inconsistent diversity (Zulfahmi, 2013).Then, molecular markers are preferable to utilize because plant DNA is stable and not affected by environmental factors.One of the most used molecular marker is Random Amplified Polymorphic DNA (RAPD).RAPD is easy and uses random primer DNA sequences as marker (Kadve et al., 2012).RAPD usually uses 10 bases short primer sequences to randomly amplify DNA genom.DNA fragmen amplification is performed using PCR machine at low annealing temperature (William et al., 1990).The DNA bands amplified from PCR can be scored to identify the genetic relationship among 15 Hibiscus genus using dendogram.DNA was extracted from a 0.1 g fresh and healthy leaf using CTAB method (Doyle and Doyle, 1990).Afterward, DNA pelette was diluted with ddH2O.DNA concetration was measured with Gene Quant Spectrophotometer, then each sample of DNA was diluted with ddH2O for temperature and primer optimization in PCR reaction.Ten optimized primers were used in this research i.e., OPA 13, OPB 18, OPC 2, OPD 2, OPD 5, OPD 7, OPD 8, OPD 18, OPD 20 and OPM 6. Optimization of annealing temperature was carried out at 35°C-45°C, then suitable temperature which produced brightest band was selected as annealing temperature.

MATERIALS AND METHODS
DNA amplification was performed with PCR (Thermal Cycler BIORAD).The touchdown method was used by reducing annealing temperature from 41°C to 37°C.The amplification reaction stage was 95°C pre-heating for 5 min; 15 cycles of 95°C denaturation for 30 sec and specific annealing temperature for 30 sec; 72°C elongation for 90 sec; and 72°C final elongation for 7 min.
Gel electrophoresis was performed in 100 volts for 55 min using 1% agarose gel in TBE buffer, visualized using UV light, and the image was captured with a digital camera.The bands were scored to form a binary data, 1 = the band was amplified, and 0 = no band.
The data was analyzed using Sequential, Agglomerative, Hierarchical, and Nested (SAHN) with Unweight pair-group method, arithmetic average (UPGMA) method for cluster analysis, in NTSYS-PC version 2.02 programs.Principal Coordinate Analysis (PCA) was performed using the same program, to illustrate the distribution of 15 Hibiscus genera.The number of specific alleles in each population, analysis of molecular variance (AMOVA), heterozygosity (H), the number of observed alleles (Na), and the number of effective alleles (Ne) were analyzed with GenAlEx 6.1.The specific alleles was determined by the appearance of a specific band in one specific population and that was not found in other populations.Analysis of molecular variance was used to explain the distribution and magnitude of genetic diversity in and/or between populations.

RESULTS AND DISCUSSION
H. rosa-sinensisis, one of the species in Hibiscus genus, the most common species found in Indonesia.Because of its flower variation, it was used as an ornamental and fence plant.Therefore, the purpose of this study was proposed to identify the diversity and genetic relationship of 15 Hibiscus genus.All of the species showed high diversity of flower morphologies as shown in figure 1.The variation of flower shapes was observed either within H. rosasinensis accessions or among other Hibiscus species Hibiscus genus consists of annual crops group such as H. cannabinus (K1) and H. sabdariffa (K2), and perennial crops group such as H.mutabilis (K3), H. syriacus (K4), H. schizopetalus (K5) and H. rosa-sinensis (R1-R10).H. rosa-sinensis accessions was separated by the flower color i.e., red flower (R1-R3), white flower (R4-R6), yellow flower (R7-R8), and orange flower (R9-R10).All Hibiscus genera were generally had similar utility for ornamental, medicinal, and fiber source plants.Genetic relationship analysis was performed in accordance to the similarity of morphology, lifespan, and utility among several types of Hibiscus.Five from ten primers showed 100% of polymorphic bands (Table 1).OPB 18, OPD 7, OPD 18, OPD 20, and OPM 6 showed 100% of polymorphic bands suggesting that 15 Hibiscus genera had high diversity.
Genetic relationship of 15 Hibiscus genera using 10 RAPD primers was identified by dendogram  Generally, the 15 Hibiscus accessions were grouped based on their population.The PCA result showed that all Hibiscus species were organized according to the flowers color (Fig. 3).Populations of the red H. rosa-sinensis are gathered into one group, as well as the orange one.While on the yellow, white, and control group, there were separated from those genotypes.K4 and K5, the perennial crops, were separated from K1 and K2, the annual crops.However, the white and yellow H. rosa-sinensis species were assembled in one group.These results suggest that all of 15 Hibiscus accessions were assembled in accordance with their flower colors.
Several variables such as observed alleles, effective alleles, heterozygosity, and Shannon index, were used to evaluate genetic diversity in a population (Pereira et al., 2015) and calculated from the three population's data (Table 2).Heterozygosity values of each population was 0.21 (control), 0.13 (red H. rosasinensis), 0.15 (white H. rosa-sinensis), 0.1 (yellow H. rosa-sinensis), and 0.06 (orange H. rosa-sinensis).These results suggest that the control group, consist of H. cannabinus, H. sabdariffa, H. mutabilis, H. syriacus and H. schizopetalus had the highest genetic diversity, whereas the orange group had the lowest genetic diversity.The observed allele value (Na) and Shannon Index (I) analysis, control group and orange group were exhibited the highest and the lowest genetic diversity, respectively.Effective allele value (Ne) is usually smaller than Na value in control population because the population consist of different kinds of Hibiscus species.However, in other population, Ne Table 3 showed the value of genetic diversity within and between populations.Diversity value between different flowers of H. rosa-sinensis flower was varied.AMOVA showed the same pattern of genetic diversity within and between populations and it was used to identify the distribution of genetic diversity within and between populations.AMOVA result (Table 3) showed that 78% of the total genetic diversity was diversed within genotype population, while the rest was diversed between populations.Distribution patterns of genetic diversity, as well as the percentage of diversity within population, was higher than between populations, which commonly found in cross-pollinated plants.
Genetic diversity within population is resulted from its sexual reproduction (Pereira et al., 2015).Then, the great diversity found in the cross pollinated crops was caused random mating.Meanwhile, population diversity is the average of genetic diversity from entire genotypes within the population.In cross-pollinated plants, although the genotypes generating each population vary, diversity value between one population and the other tend to be small, so the diversity between populations is small.This is in accordance with Hamrick et al. (1992), that wood species with cross-breeding system and seed dispersal caused by wind or animal will maintain more variation within species and population than other species, but the diversity between populations is smaller.

CONCLUSIONS
The molecular marker is a good marker to reveal the diversity in a population.Highest heterozygosity value in control group genotypes showed that there was a high diversity in control plants.Based on dendogram result, H. rosa-sinensis had a close genetic relationship with H. schizopetalus.Based on the PCA result, the grouping in control plants was assembled according to their growth cycles (perrenial and annual), while in H. rosa-sinensis the grouping was based on the flowers color.H. rosa-sinensis is cross pollinated plants because the stigma position is higher than anthers, and the varians within population of H. rosa-sinensis was higher than among population.

Table 1 .
Polymorphic Bands 15 Hibiscus Genus in 10 Primer RAPD R2 and R3 genotypes with the similarity coefficient was 0.72.Then B.2.2.2 consisted of 6 genotypes where R9 and R10 have the closest genetic relationship (similarity coefficient 0.78) compared with other genotypes (R7 and R8 with similarity coefficient 0.65, and R4 and R5 with similarity coefficient 0.73).H. rosa-sinensis group was tend to gather, separated from the other Hibiscus species.