Abstract: Background and Objective: Moringa (Moringa oleifera Lam.) which also known as drumstick tree has been widely used for functional food in many countries and has spread out to almost all over the world including Indonesia. This study aimed to identify the genetic diversity of 30 genotypes from 10 populations of Moringa plants originating from several islands in Indonesia using SRAP molecular markers in combination with observations of morphological characters. Materials and Methods: In this experiment, the genetic diversity of Moringa from several islands in Indonesia was evaluated using Sequence Related Amplified Polymorphism (SRAP) molecular markers as well as morphological characters. A total of 30 genotypes of Moringa from 10 different islands in Indonesia were planted in 10 kg of polybags for 3-7 months to observe their molecular as well as morphological characters. For molecular analysis, 10 selected primers of SRAP were used. Results: The 70 polymorphic bands from 86 total bands (81.40%) were obtained and Polymorphic Information Content (PIC) values were 0.36 on average. The UPGMA analysis divided 10 accessions into 2 main groups which Java accession was grouped alone and separated from other accessions. The principle component analysis based on morphological characters divided them into 4 groups with Java accession consistently separated from others. Conclusion: Based on molecular and morphological analysis, accession of Java was the most distinct Indonesian Moringa accession suggesting the narrow distribution of this accession than others. Leaflets number, tuberous root number and color were among the most variables that influenced the distinction of Moringa accession from Indonesian archipelago.
INTRODUCTION
Moringa oleifera Lam. or drumstick tree is a fast-growing tree with an average height can reach up to 12 m has a fragile trunk with a gray-whitish bark. The plant has compound leaves arranged in a spiral and consist of tens to hundreds of oblong or elliptical leaflets with small size, 1.2-2.0 cm long and 0.6-1.0 cm wide1. Moringa plant is known to have high nutritional content and bioactive compounds and therefore this plant has been widely used as food, medicine and even cosmetics2. This plant is indigenous to Himalayan foothill covering Pakistan, India, Nepal and Bangladesh1 and has currently spread almost throughout the world, especially in the tropical region, including Indonesia3,4.
Several studies on genetic diversity of moringa plants have been conducted including Silva et al.5 using Moringa from Brazil, Shahzad and Khan6 and Ganesan et al.7 using Moringa from Pakistan, Ojuederie et al.8 using Moringa from Nigeria, Rajalakshmi et al.9 and Ravi et al.10 using Moringa from India and Kleden et al.11 using Moringa from Indonesia. Meanwhile, the genetic diversity studies of Moringa plants from the Indonesian archipelago are still very limited. Even though they use Moringa from Indonesia, Kleden et al.11 only used Moringa from one island, namely Timor Island. So far, no one has studied the genetic diversity of Moringa plants from various islands in Indonesia. Information about the genetic diversity of Moringa plants is important for further development, breeding and cultivation on an industrial scale.
Observation of morphological characters can be carried out on vegetative organs and / or plant generative organs, as has been done by Salem et al.12 on wheat, Cortese et al.13 on Switchgrass plants and Ebrahimi et al.14 on Persian Walnut (Juglan regia L.). Even, the minimum morphological characters of Moringa plants for genetic diversity studies have been formulated by Santhoshkumar et al.15. In addition to morphological analysis, plant genetic diversity can be studied using molecular approaches. Molecular markers that have been commonly used in plant genetic diversity study include RAPD (Random Amplified Polymorphic DNA)5,11,16,17, ISSR (Inter Simple Sequence Repeat)18-21, AFLP (Amplified Fragment Length Polymorphism)22-25, SSR (Simple Sequence Repeat)6,7,9 and SRAP (Sequence Related Amplified Polymorphism)26-29. From those molecular markers, SRAP is a molecular marker that has never been used to study the genetic diversity of Moringa plants, in spite of some advantages over other molecular markers. The RAPD and ISSR are reported to have low levels of accuracy, reproducibility and the ability to detect polymorphism, whereas AFLP is expensive and has more complicated procedure. The SRAP requires a relatively low cost with simpler techniques but has high accuracy, reproducibility and the ability to detect polymorphism30. SRAP has also been reported to be effectively successful in identifying the genetic diversity of tree plants such as bamboo26, Dalbergia29 and Ramin31.
The purpose of this study was to identify the genetic diversity of 30 genotypes from 10 populations of Moringa plants originating from several islands in Indonesia using SRAP molecular markers in combination with observations of morphological characters.
MATERIALS AND METHODS
Time and location: The experiment was carried out from 6th April to 25th September, 2020, in the Glass House and the Laboratory of Plant Molecular Systematics and Laboratory of Genetic and Plant Breeding, Research Centre for Biology, Indonesian Institute of Sciences, Bogor, West Java, Indonesia.
Plant materials: The plant materials were obtained from Research Center for Biology, Indonesian Institute of Sciences consisted of 30 Moringa genotypes from 10 accessions/ population collected from 10 different islands in Indonesian archipelago, namely Sumatra (Deli Serdang), Java (Bogor), Madura (Sumenep), Bali (Tabanan), Lombok (Kuta-Mandalika), Sumbawa (North Moyo), Sumba (Southwest Sumba), Borneo (Mempawah), Celebes (Enrekang) and Papua (Jayapura) (Fig. 1). The 10 accessions of Moringa seeds were washed in running water and then soaked in clean water for 1 hr to induce water imbibition. The seeds were then soaked in solution contained fungicides and bactericides 2 g L–1 water for 30 min before drained. A total of 3 seeds for each accession were planted on a 35×45 cm polybag containing a mixture of soil, manure and roasted husk with a ratio of 2:1:1 (v/v/v) for a total weight of 10 kg. The humidity of the media was maintained by watering every 2 days. The plants were grown for 7 months to provide the adequate samples for molecular analysis as well as morphological observation.
Molecular analysis: Analysis of genetic diversity was carried out using Sequence-Related Amplified Polymorphism (SRAP) molecular markers. DNA was isolated from young leaf samples using Genomic DNA Mini Kit (Plant) from Gene Aid. Genomic DNA amplification of Moringa plants was performed using 10 SRAP primers combinations (Table 1) which had been screened before from 25 pairs of primers developed by Li and Quiros32. The PCR reaction consisted of 1×PCR master mix (Promega), 10 ng templates DNA, 2 μM for each primer with a total volume of 15 μL. The PCR amplification was performed at the following optimum conditions: 94°C for 5 min, 50°C for 45 sec and 72°C for 2 min.
| Fig. 1: | Map of the location of plant material collection 1: Sumatera, 2: Java, 3: Madura, 4: Bali, 5: Lombok, 6: Sumbawa, 7: Sumba, 8: Borneo, 9: Celebes and 10: Papua |
| Table 1: List of SRAP primer pairs and its sequences used in the study | |
| Primer pairs | Sequences |
| me 1F and em 1R | TGAGTCCAAACCCGATA and GACTGCGTACGAATTAAT |
| me 1F and em 4R | TGAGTCCAAACCCGATA and GACTGCGTACGAATTTGA |
| me 1F and em 5R | TGAGTCCAAACCCGATA and GACTGCGCACGAATTGCA |
| me 2F and em 1R | TGAGTCCAAACCGGAGC and GACTGCGTACGAATTAAT |
| me 2F and em 2R | TGAGTCCAAACCGGAGC and GACTGCGTACGAATTTGC |
| me 2F and em 4R | TGAGTCCAAACCGGAGC and GACTGCGTACGAATTTGA |
| me 3F and em 1R | TGAGTCCAAACCGGAAT and GACTGCGTACGAATTAAT |
| me 3F and em 2R | TGAGTCCAAACCGGAAT and GACTGCGTACGAATTTGC |
| me 3F and em 3R | TGAGTCCAAACCGGAAT and GACTGCGTACGAATTGAC |
| me 3F and em 4R | TGAGTCCAAACCGGAAT and GACTGCGTACGAATTTGA |
| Table 2: Description keys of Moringa oleifera qualitative characters | |
| Characters | Descriptions |
| Leaflets shape | Elliptical, Oblong7 |
| Leaflets color | Based on RHS mini color chart |
| Stem bark shape | Striped, warty, spotting36 |
| Stem bark color | Based on RHS mini color chart |
| Tuberous root color | Based on RHS mini color chart |
The reaction was stopped by extension at 72°C for 5 min. Amplification was visualized with 1.5% agarose gel and stained with GelRed (Biotium) before being photographed using the gel documentation system (Atto Bioinstrument). The scoring for each sample was carried out by observing the results of electrophoresis, where a score of 1 was given for the present of band and 0 for the absent of band.
AMOVA analysis: AMOVA analysis and the calculation of the genetic distance of accessions were carried out using GenAlEx software13. UPGMA grouping analysis based on a genetic distance matrix was carried out using the Mega X software33. Polymorphism Information Content (PIC) was calculated using Microsoft Excel with the formula used by De Riek et al.34. PCA clustering analysis was performed using MetaboAnalyst 4.035. Quantitative morphological data were analyzed with ANOVA using SPSS for Windows version 16 software at α = 0.05. Post hoc was carried out with Duncan Multiple Ranges Test (DMRT) at α = 0.05.
Morphological characters analysis: Morphological characters were observed by analyzing the leaves, stems and roots, qualitatively and quantitatively. The characters and the descriptions of qualitative observations are presented in Table 2. The quantitative characters were observed by measuring plant height, stem diameter, canopy diameter, compound leaves number and sizes, leaflets number and area, tuberous root number, diameter and length. Plant height was measured from the soil surface to the top of the youngest shoot. Stem diameter was observed at a height of 5 cm from the surface of the medium. Canopy diameter was measured on the widest part of the canopy from 2 perpendicular sides. The number of compound leaves was calculated based on the number of leaves still attached to an individual plant. The length and width of compound leaves as well as the number and the area of leaflets were measured to the 4th compound leaves calculated from the shoot apex. The length of compound leaves was measured from the base of the petiole to the tip of the leaves, while the width of the leaves was measured at the widest part of the leaves. The leaflets number was counted manually from one compound leaf. The leaflets area was calculated by measuring 5 leaflets taken randomly from each 4th compound leaf, scanned and calculated using ImageJ 1.47v software. Observation of the number and the length of roots were carried out by counting the tuberous roots and by measuring the base of the tuberous root until the longest part of root tip. The tuberous root diameter was measured on the largest part. The observations of the shoot were carried out at the age of 3 months after planting (map), while the observations of the roots were carried out with destructive measurement at the age of 7 maps. The observation of tuberous root color was carried out using the RHS mini color chart.
RESULTS
Molecular analysis of Moringa: PCR analysis using 10 primer pairs of 10 accessions of Moringa plants obtained an average of 8.6 amplified fragments. As presented in Fig. 2, the primer pairs that produced the most amplified fragments were me2F and em1R (Fig. 2a), while the least was me1F and em4R (Fig. 2b). The primer pairs that produced the highest polymorphisms were me1F and em1R (100%) (Fig. 2c), while the lowest was me2F and em2R (57%) (Fig. 2d). Overall, the average polymorphic fragment was 7.0 or 81.40%. The PIC values ranged from 0.26-0.45 with an average of 0.36. The amplified fragment sizes ranged from 150-2900 base pairs (bp) (Table 3).
The AMOVA analysis showed that the percentage of genetic variation among populations was higher than that within population, i.e.: 54 and 46% respectively (Fig. 3). It means that the genetic variation of 10 accessions of Moringa from various islands in Indonesia was much influenced by the origin of the accessions/populations, suggesting that Moringa plants from various islands in Indonesia have high genetic differences.
| Fig. 2(a-d): | SRAP profile of 10 accessions of Moringa oleifera using (a) me2F and em1R, (b) me1F and em4R, (c) me1F and em1R and (d) me2F and em2R primer pairs combinations First lane from the left bands was 100 bp DNA ladder, the second to the eleventh lane were the accession of Sumatera, Java, Madura, Bali, Lombok, Sumbawa, Sumba, Borneo, Celebes and Papua |
| Table 3: | List of primer pairs, number of amplified fragments, number of polymorphic fragments, polymorphic information content (PIC) and fragment size range of 10 Moringa oleifera accessions |
Numbers of |
Numbers of |
Size |
||
| Primer pairs | amplified fragments |
polymorphic fragments |
PIC |
range (bp) |
| me 1F and em 1R | 11 |
11 |
0.34 |
150-2800 |
| me 1F and em 4R | 4 |
3 |
0.34 |
180-2500 |
| me 1F and em 5R | 8 |
6 |
0.38 |
200-2500 |
| me 2F and em 1R | 15 |
14 |
0.26 |
150-2900 |
| me 2F and em 2R | 7 |
4 |
0.40 |
190-2500 |
| me 2F and em 4R | 8 |
5 |
0.45 |
200-2500 |
| me 3F and em 1R | 7 |
6 |
0.41 |
300-1200 |
| me 3F and em 2R | 12 |
11 |
0.24 |
150-2000 |
| me 3F and em 3R | 6 |
5 |
0.42 |
200-1000 |
| me 3F and em 4R | 8 |
5 |
0.35 |
400-2900 |
| Total | 86 |
70 |
||
| Fig. 3: | AMOVA based on SRAP data for 30 genotypes from 10 accessions of Moringa oleifera |
The UPGMA dendrogram showed that the 10 accessions of Moringa were divided into 2 groups. The Java accessions (J) were grouped in a sole group, while the other 9 accessions were grouped into the other (Fig. 4a). The second group was divided into 3 subgroups. The first subgroup was Madura (M) and it was the nearest to Java accession (J). In the second subgroup, Sumatera accessions (Sm) were grouped together with Bali (B) and separated from the third which contained 6 accessions. In the third subgroup, Lombok (L) was grouped alone, while Papua (P) was grouped with Celebes (Si), Sumbawa (Sbw) grouped with Sumba (S) and Borneo (K). The clustering analysis using Principle Component Analysis (PCA) showed the same pattern as well, where Java accessions (J) were grouped in sole group (the first group) and other accessions belong to other groups (the second group). The second group was divided into 3 subgroups. The first subgroup includes Sumbawa (Sbw) and Lombok (L), the second subgroup includes Sumatera (Sm) and Bali (B) and the third subgroup includes Madura (M), Sumba (S), Borneo (K), Celebes (Si) and Papua (P) (Fig. 4b).
Morphological analysis
Qualitative characters: The qualitative character analysis of the 10 Moringa accessions showed differences in stem bark shape, stem bark color and the tuberous root color, while the leaflets shape and color were not different (Table 4). Borneo accession had a striped bark shape, while other accessions had a uniform bark shape, namely warty. The stem bark color of the 10 accessions was divided into 2 groups, brown color for Sumatra (Sm), Madura (M), Lombok (L), Borneo (K), Celebes (Si) and green color for Java (J), Bali (B), Sumbawa (Sbw), Sumba (S) and Papua (P). Tuberous root color was divided into three grades of yellow, i.e., moderate orange yellow 164 B for Sumatra (Sm), Bali (B), Lombok (L), Sumbawa (Sbw) and Sumba (S), moderate orange yellow 164 C for Madura (M) and Borneo (K) and pale yellow 164 D for Java (J), Celebes (Si) and Papua (P).
Quantitative characters: The analysis of ANOVA to morphological characters showed that plant height, stem diameter, canopy diameter and number of taproots were significantly different among the accessions. Accession of Madura (M) was the tallest even though not significantly different from Java (J) and Celebes (Si) accessions. The shortest accessions were Sumba (S) and Borneo (K) (Fig. 5a). The Java accession (J) had the largest trunk diameter, while Lombok (L), Sumbawa (Sbw), Sumba (S) and Borneo (K) had the smallest (Fig. 5b). The biggest diameter of the canopy was found in Java accession (J), while the smallest was in Bali (B) (Fig. 5c). The highest number of tuberous roots was observed in Java (J) and Celebes (Si) accessions, while the lowest was observed in Sumatra accessions (Sm) (Fig. 5d).
| Fig. 4: | (a) UPGMA dendrogram of 10 accessions of Moringa oleifera based on Nei’s genetic distance and (b) PCA based on SRAP data using Metaboanalyst 4.0 Sm: Sumatera, J: Java, M: Madura, B: Bali, L: Lombok, Sbw: Sumbawa, S: Sumba, K: Borneo, Si: Celebes, P: Papua |
| Table 4: Qualitative characters of 10 Moringa oleifera accessions | |||||
| Accessions | Leaflet shape | Leaflet color | Stem bark shape | Stem bark color | Tuberous root color |
| Sumatera | Elliptical | Moderate olive green A | Warty | Brown 47 RHS 199C | Moderate orange yellow 164 B |
| Java | Elliptical | Moderate olive green A | Warty | Green 42 RHS 194A | Pale yellow 164 D |
| Madura | Elliptical | Moderate olive green A | Warty | Brown 47 RHS 199C | Moderate orange yellow 164 C |
| Bali | Elliptical | Moderate olive green A | Warty | Green 42 RHS 194A | Moderate orange yellow 164 B |
| Lombok | Elliptical | Moderate olive green A | Warty | Brown 47 RHS 199C | Moderate orange yellow 164 B |
| Sumbawa | Elliptical | Moderate olive green A | Warty | Green 42 RHS 194A | Moderate orange yellow 164 B |
| Sumba | Elliptical | Moderate olive green A | Warty | Green 42 RHS 194A | Moderate orange yellow 164 B |
| Borneo | Elliptical | Moderate olive green A | Striped | Brown 47 RHS 199C | Moderate orange yellow 164 C |
| Celebes | Elliptical | Moderate olive green A | Warty | Brown 47 RHS 199C | Pale yellow 164 D |
| Papua | Elliptical | Moderate olive green A | Warty | Green 42 RHS 194A | Pale yellow 164 D |
| Fig. 5(a-i): | Moringa oleifera accessions of (a) Plant height, (b) Stem diameter, (c) Canopy diameter, (d) Tuberous root number, (e) Compound leaves number, (f) Compound leaves length, (g) Compound leaves width, (h) Leaflets number and (i) Leaflet area Sm: Sumatera, J: Java, M: Madura, B: Bali, L: Lombok, Sbw: Sumbawa, S: Sumba, K: Borneo, Si: Celebes, P: Papua. Different letters indicate significant differences at p<0.05 DMRT |
| Fig. 6(a-b): | The analysis of (a) PCA-2D and (b) PCA-Biplot of 10 M. oleifera accessions based on morphological data, using Metaboanalyst 4.0 program Sm: Sumatera, J: Java, M: Madura, B: Bali, L: Lombok, Sbw: Sumbawa, S: Sumba, K: Borneo, Si: Celebes, P: Papua |
Based on all those parameters, Java accessions (J) was the most different from others with the specific and distinct characters including relatively higher plant, biggest stem diameter, widest canopy and the most tuberous roots. Based on the quantitative character of the leaves, Bali accession (B) had the largest compound leaves, but not significantly different from Java accession (J), while the accession that have the least compound leaves was Borneo (K) (Fig. 5e). The accessions with the longest and widest compound leaves were Java (J) and Celebes (Si) (Fig. 5f-g). Among all the accessions, Java (J) had the highest leaflets number (Fig. 5h), even though the size of leaflet was smallest (Fig. 5i).
The PCA analysis carried out using both quantitative and qualitative morphological characters showed that the 10 accessions of Moringa were divided into 4 groups, i.e., Java (J) in the first group, Papua (P) and Celebes (Si) in the second group, Borneo (K) in the third group and the other accession namely Madura (M), Bali (B), Lombok (L), Sumbawa (Sbw) and Sumba (S) gathered in the fourth group (Fig. 6a). Biplot PCA analysis showed that the number of tuberous roots, tuberous root colour and the number of leaflets were the three main variables that caused accession of Java (J) different from other accessions (Fig. 6b).
DISCUSSION
Molecular analysis using SRAP molecular markers effectively identified the genetic diversity of 10 Moringa accessions from various islands in Indonesia (Fig. 2 and Table 3). The percentage of polymorphisms obtained was 81.40% which indicates a fairly high genetic diversity of Moringa. Ravi et al.10 stated that the number of polymorphic loci or the percentage of polymorphisms is a good indicator of Moringa genetic diversity. The percentage of polymorphisms in Moringa genetic diversity reported from several previous studies was lower than this result, such as 66.5% in Moringa from India, Malawi and Kenya using AFLP markers36, 55.9% in Moringa from Timor Island, Indonesia11, 48.68% in Moringa from India19 and 62% in Moringa from the United States5 using RAPD markers, 48.57% using ISSR markers and 40.00% using cytochrome p450 in Moringa from India19. There were also some reports of equivalent or even slightly higher percentage of polymorphisms, such as Ojuederie et al.8 in Moringa from Nigeria using RAPD markers which was 81.50%, Ganesan et al.7 in Moringa from India with SSR markers (82.86%) and Ravi et al.10 in Moringa from India using cytochrome p450 which was much higher (88.25%). The higher percent polymorphism resulted in this experiment indicated the diversity and uniqueness of Moringa collected from different islands separated by seas in the Indonesian archipelago. This was confirmed by the result of AMOVA analysis which shows that the genetic variation among islands was greater than within islands (Fig. 3).
The UPGMA clustering analysis based on the genetic distance matrix among populations and PCA based on the SRAP data showed that the accession of Java (J) was the most different accession compared to other accessions (Fig. 4a). The data of PCA analysis generated from morphological characters also separated Java accession (J) from other accessions (Fig. 6a). Even though in this analysis, the 10 accessions were divided into 4 groups, but Java (J) accession was still separated into different group far from others. Java accession (J) had more superior morphological characters such as stem diameter, canopy diameter, number of compound leaves and leaflets, number of tuberous roots, even though it had smallest size of leaflets. This accession also had brightest color of tuberous roots. These characters made Java accession (J) to be relatively far apart from other accessions in the PCA analysis. Among those characters, leaflet number, tuberous root number and color were among the most variables that influenced the distinction of Moringa accession of Java (J) (Fig. 6b). The distinctive of Java accession (J) compared to other accessions indicates a narrow distribution of this accession, while 9 others which had relatively higher similarity which indicated a wider distribution. Chen et al.37 stated that the distribution of plants is influenced by internal factors which refer to the characters of the plant itself and external factors including the role of human intervention. Shahzad et al.6 reported that M. oleifera accessions from 9 different countries (India, Senegal, Tanzania, Mozambique, Zimbabwe, Florida, Belize, Mexico and Haiti) had low genetic diversity and had high similarities to one accession from Pakistan (Punjab accession). Based on this, they assumed that the accessions from 9 different countries originated from Punjab and then spread to these 9 countries due to human intervention through introductions by traders, expatriate, immigrants and even by colonialists in the past. Human’s intervention also influences the distribution of plants through plant selection activities for breeding and cultivation which is focused on their superior characteristics. Plants with characters considered less interesting tend to be abandoned and usually obtain lower human intervention. Consequently, the distribution of these plants occurs naturally and relatively narrow.
The smaller leaflet size of Java accession (J) may be one reason that caused this accession has less attention by breeders and cultivators, so it tends to be neglected since the leaves are the most widely used organ harvested from this plant2,38,39. Morphological characters of a plant are commonly used as the basis for plant selection efforts in breeding and cultivation programs. Some common morphological characters that have been used for breeding selection in many cultivated species include the size, shape, leaf colour, fruit size and weight in almond plants40, the number, size and weight of pods and seeds in cowpeas41,42, the size and weight of fruit in chili43, the size and head yield in cabbage44, size and color of leaves in Cleome gynandra L.45 and the leaf size and weight in amaranth vegetables46. The local community traditions probably also have determined the narrow distribution of Java accession (J). The people of West Java where the accession was collected generally use the Moringa plant only as an ingredient for bathing corpses and not for food and therefore this accession is not intensively cultivated in this area. This is probably the reason that the introductions of Moringa genotypes from outside islands are rarely found.
CONCLUSION
It can be concluded that SRAP molecular markers can be effectively used to detect the genetic diversity of M. oleifera plants by obtaining 81.40% polymorphic bands which indicated higher genetic diversity of Indonesian Moringa. The accession of Java was the most distinct accession than others indicated the narrow distribution of this accession due to probably lower human intervention. The leaflets number, the number of tuberous root and the color of tuberous root were among the most influential variables which caused distinction of the accession.
SIGNIFICANCE STATEMENT
This study discovered that there were a considerable variation of Drumstick (Moringa oleifera Lam.) accessions collected from 10 islands in Indonesia based on morphological and molecular analysis that has never been reported before, which can be beneficial for researchers and breeders in utilizing the accession for further development. This study will help the researchers to uncover the critical areas especially the differences of secondary metabolic content among the accessions that many researchers were not able to explore. Thus a new theory on the diversity of drumstick tree based on bioactive compounds may be arrived at the near future.
ACKNOWLEDGMENT
This research was funded by The Ministry of Research and Technology/National Research and Innovation Agency through Doctoral Dissertation Research Program, with the Grant number of 1/E1/KP.PTNBH/2020. We also would like to appreciate all staff of Plant Molecular Systematics Laboratory and Genetic and Plant Breeding Laboratory of Research Center for Biology, Indonesian Institute of Sciences, especially Kusuma Dewi Sri Yulita and Seniwati for their time to discuss the SRAP molecular markers and Susila and Erlin for their technical assistance during this research.