Indonesian Journal of Agricultural Science Vol. 20 No. 1 June 2019: 01–08

DOI: http//dx.doi.org/10.21082/ijas.v.20.n1.2019.p.01–08

GENETIC VARIABILITY OF INDONESIAN PAPAYA ACCESSIONS AS REVEALED BY RANDOM AMPLIFIED POLYMORPHIC DNA AND MORPHOLOGICAL

CHARACTERIZATION

Keragaman Genetik Aksesi Pepaya Indonesia Hasil Karakterisasi Marka Random Amplified Polimorphic DNA dan Karakter Morfologi

Riry Prihatini, Tri Budiyanti, and Noflindawati

Indonesian Tropical Fruit Research InstituteJalan Raya Solok-Aripan km 8, Solok 27301, West Sumatera, Indonesia

Phone: +62 75520137; Fax: +62 75520592* Correspondence author: riry_silva@yahoo.com

Submitted 9 October 2018 ; Revised 21 Januari 2019 ; Accepted 11 May 2019

ABSTRACT

Diverse papaya (Carica sp.) accessions are found in many regions in Indonesia, but their genetic diversity have not yet been studied. Random Amplified Polymorphic DNA (RAPD) is a simple yet accurate method that can be used to examine the genetic diversity of papaya. The study aimed to examine the genetic diversity of Indonesian papaya accessions using RAPD markers and morphological characters. The RAPD was applied on 23 papaya accessions using 30 primers. The appearing bands were further analyzed with the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and Principal Component Analysis (PCA). The molecular results were then compared to the fruit morphological data, including fruit shape, size, flesh color, texture, and flavor. The RAPD analysis revealed that the 23 papaya accessions clustered into six main clades with Dice-Sorensen coefficient similarity ranged from 0.71 to 0.98. The first group consisted of 11 accessions, including both the hybrids and local accessions. The second group consisted of eight accessions especially six Indonesian hybrids, a Mexican Hybrid and a Hawaiian hybrid. The other four groups had a single member namely Sicincin Panjang, Lokal Sumani, Cariso, and Carica. The molecular grouping, however, did not align with the fruit character grouping. Overall, it was implied that the Indonesian papaya accessions were genetically narrow, of which some accessions were closely related to Hawaiian and Mexican accessions. These results can be used as a reference on papaya crossbreeding program in Indonesia.

[Keywords: Carica sp., genetic diversity, morphological characters, PCA, RAPD]

ABSTRAK

Beragam aksesi pepaya (Carica sp.) ditemukan di berbagai daerah di Indonesia, namun keragaman genetiknya belum diketahui. Rapid Amplified Polymorphic DNA (RAPD) merupakan metode sederhana namun akurat untuk mengetahui keragaman genetik pepaya Indonesia. Penelitian ini bertujuan untuk mempelajari keragaman genetik aksesi pepaya Indonesia menggunakan marka RAPD dan karakter morfologis. RAPD dilakukan pada 23 aksesi pepaya dengan menggunakan 30 primer. Pita yang dihasilkan dianalisis lebih lanjut dengan Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dan Principal

Component Analysis (PCA). Hasil analisis molekuler kemudian dibandingkan dengan data morfologi buah, yang meliputi ukuran, warna daging, tekstur, dan rasa. Hasil analisis RAPD menunjukkan bahwa 23 aksesi pepaya Indonesia mengelompok menjadi enam kelompok utama dengan koefisien kesamaan Dice-Sorensen berkisar 0,71–0,98. Kelompok pertama terdiri atas 11 aksesi, termasuk aksesi lokal dan hibrida. Kelompok kedua terdiri atas delapan aksesi, yaitu enam aksesi pepaya hibrida Indonesia, satu hibrida asal Meksiko, dan satu hibrida asal Hawaii. Sementara empat kelompok lainnya masing-masing memiliki satu anggota, yaitu Sicincin Panjang, Lokal Sumani, Cariso, dan Carica. Namun, pengelompokan molekuler tersebut tidak sejalan dengan pengelompokan berdasarkan karakter buah. Secara keseluruhan, hasil analisis menunjukkan bahwa aksesi pepaya Indonesia memiliki keragaman genetik yang sempit, termasuk beberapa aksesi yang berkerabat dekat dengan aksesi asal Meksiko dan Hawaii. Hasil penelitian ini dapat menjadi referensi dalam program pemuliaan pepaya di Indonesia.

[Kata Kunci: Carica sp., keragaman genetik, karakter morfologis, PCA, RAPD]

INTRODUCTION

Papaya is originated from three centers, called as Mesopotamic, Mesoamerican, and North Chinese. Since its wide adaptation capacity, the plant has spread on to almost tropical and subtropical regions. In Indonesia, papaya is cultivated mainly for the fruits, yet the latex, seeds, and leaves are commonly used as traditional remedies. Papaya can be cultivated in almost every part of Indonesia with diverse fruit shape, size, and color. A study reported that Indonesian papaya morphological caharacters varied pertaining to vegetative and generative characters (Suketi et al. 2010). Similar study showed a wide significant variation within the cultivated papaya in Kenya, including fruit characters, petiole length, and leaf size which were found to be highly correlated among them (Asudi et al. 2010). Costa Rica’s papaya

2 Indonesian Journal of Agricultural Science Vol. 20 No. 1 June 2019: 01–08

had a wide variation in the morphological characters that were not relevant to the genetic analysis (Brown et al. 2012). In addition, Ocampo et al. (2006) found that the sexual type of papaya influenced the leaf and flower characteristics, as a result of environmental effect.

Papaya accession classification should be done using not only morphological characters but also molecular markers. The common method to access plant genetic information is Random Amplified Polymorphic DNA (RAPD), which is advantages in rapid, relatively easy and simple technique, thus can be operated in a undeveloped laboratory with minimum equipments. RAPD analysis is applicable in confirmation of genetic linkage and diversity (Nejatzadeh-Barandozi et al. 2012), somaclonal variation (Dey et al. 2015), and sex determination in seedling (Vaidya and Naik 2014).

Papaya genetic diversity had been widely studied, included the use of microsatellite markers to assess polymorphism. Other studies had focused on the relationship among papaya and its relatives (Carvalho and Renner 2012), also among papaya local germplasms (Ocampo et al. 2006; O. Aikpokpodion 2012; Madarbokus and Ranghoo-Sanmukhiya 2012, Saran et al. 2015). However, to date, there is a little information on the study of Indonesian papaya molecular diversity. The study aimed to determine the diversity of Indonesian papaya accessions based on RAPD markers and morhological characters.

MATERIALS AND METHODS

Plant Materials

A total of 22 papaya accessions collected and managed by the Indonesian Fruits Research Institute under the Indonesian Agency for Agricultural Research and Development (IAARD) were used in this study. The genetic materials included 12 hybrids, 8 local accessions, and 2 introduced accessions (Table 1). These accessions have been priorities in the IAARD papaya breeding program.

Morphological Characterization

A number of morphological characters of fruits were observed to support the molecular analysis. The investigated morphological characters were the principal characters such as fruit shape and size, flesh color and texture, and flavor which were to be highly correlated to the papaya diversity (Ocampo et al. 2006; Asudi et al. 2010).

Extraction of Genomic DNA

The DNA was extracted from young healthy leaves using the modified CTAB method (Doyle and Doyle 1987). A total of 100 mg leaves were ground with 1.5 ml extraction buffer, 1% β-mercaptoethanol, and 10 mg PVP-10. The samples were then transferred into a 2 ml centrifuge tube and incubated at 65 ⁰C for 60 minutes. Proteins were degraded three times with 500 ml chloroform : soamyl-alcohol (24:1, v/v) and centrifuged at 12,000 rpm for 10 minutes. The DNA was precipitated with the addition of 500 ml cold iso-propanol, whereas the RNA was degraded with 2 mg ml-1 RNAse. The DNA pellets were air dried, rinsed with 70% ethanol, and dissolved in 50 ml TE buffer. The DNA was quantified using nano-drop spectrophotometer. The DNA solution were kept in -20 ⁰C freezer prior to PCR analysis.

PCR Amplification

RAPD primers were initially screened from a total of 30 primers (Table 2) to find the suitable primers for revealing molecular diversity on individual level. The primers were selected using five samples of papaya leaves. The selected RAPD primers were then used for genotyping of

Table 1. A list of 23 papaya accessions used in this study.

Accesssions Genetic background Breeding statusBTK3 Cross breeding Third filial

BT2 Cross breeding Fifth filial

BT3 Cross breeding Fifth filial

BT4 Cross breeding Fifth filial1XD Cross breeding Fifth filial2X1 Cross breeding HybridsG10 Cross breeding HybridsG15 Cross breeding Hybrids

G18 Cross breeding HybridsG28 Local acession HybridsG30 Local acession HybridsDampit Local acession HybridsMerah Delima Cross breeding Fifth filialSolinda Cross breeding Fifth filialCarisya Unknown UnknownCariso Unknown UnknownDaun Jarak Local acession Local acessionTangkai Ungu Local acession Local acessionSicincing Panjang Local acession Local acessionLokal Sumani Local acession Local acessionSunrise Introduced accessionRed Lady Introduced accessionCarica Local acession

3Genetic variability of Indonesian papaya accessions … (Riry Prihatini et al.)

23 papaya accessions. PCR amplification was performed based on previous protocol (Prihatini et al. 2016) with the annealing temperatures adjusted according to each primer as presented in Table 2.

Data Analysis

The binary data of the banding patterns produced by each primer were further analyzed using computer software. A dendrogram of total accessions based on RAPD polymorphism was generated using the Dice-Sorensen coefficient of similarity together with genetic distance among groups and

the Unweighted Pair Group Method with Arithmatic Mean (UPGMA). Relationship among the hybrids and parents was investigated using Principal Coordinate Analysis based on Eigen value. All of the calculation was conducted using the NTSYSpc 2.10x software (Rohlf 2004), whereas the bootstrap analysis was performed with the Winboot software (Yap and Nelson 1996).

The polymorphic information content (PIC) value was calculated for each marker based on formula of PIC = 2fi(1-fi), where fi is the frequency of the amplified alleles and (1-fi) is the null alleles. The average of PIC value was noted as average heterozygosity (Hav). The effective multiplex ratio (EMR) is the ratio of polymorphic and total amplified bands, whereas the marker index (MI) was calculated by multiplying Hav and EMR values (Roldán-Ruiz et al. 2000; Powell et al. 1996). The genetic diversity index was estimated as the proportion of polymorphic loci across the genome (Nei 1977).

RESULTS AND DISCUSSION

Fruit Morphological Characters

As a part of previous papaya breeding studies, several hybrids have been collected, both for documentation and collection for future research. The collection was then expanded to local and introduced accessions. Fruit characteristics of 23 papaya accessions in our study were presented in Table 3 and Figure 1. The fruit shape was diverse such as pear, globular, elongate, and club shape, whereas the size varied from small (√) to very big (√√√√√). Similarly, the flesh fruit color varied from yellow, orange, orange-reddish, reddish, to red, with differed textures, crunch and soft. In addition, the fruit flavor ranged from slightly sweet (+) to very sweet (+++). In general, the Indonesian papaya tend to have a bigger size (more than 2000 g) than the introducing ones.

Most of the hybrids were developed by IAARD and have been studied since 1996. The hybrids were evaluated to obtain the superior variety to be released to the public. An example for the hybrids is Merah Delima that has been cultivated in many farmlands and produced satisfactory yield. The other commercial hybrids were Carisya and Cariso which were released by Bogor Agricultural University prior to the Merah Delima.

The local accessions included in study had specific characters, in particular superior size (for G30, Sicincin Panjang, and Lokal Sumani), purple color petiole (Tangkai Ungu), unique leaves (Daun Jarak), and unique taste (Carica). Notable, some of these accesssions have been cultivated by local farmers, either as backyard plant or an orchard. Considering the specific characters

Table 2. The 30 RAPD primers used in this study.

Primer Sequence% GC content

Annealing temperature (oC)

RAPD1 GCGGGTGGAA 70 36

RAPD2 GTT TCGCTCC 60 36

RAPD3 GTAGACCC T 50 36

RAPD4 AAGAGCCCGT 60 36

RAPD5 AACGCGCAAC 60 36

RAPD6 CCCGTCAGCA 70 36

OPA01 CAGGCCCTTC 70 36

OPA02 TGCCGAGCTG 70 32

OPA11 CAATCGCCGT 60 36

OPA14 TCTGTGCTGG 60 36

OPC01 TTCGAGCCAG 60 35

OPC04 CCGCATCTAC 70 35

OPN09 TGCCGGCTTG 70 36

OPY15 AGTCGCCCTT 60 36

OPA03 AGTCAGCCAC 60 32

OPA07 GAAACGGG TG 60 34

OPA10 GTGATCGCAG 60 32

OPA13 CAGCACCCAC 70 35

OPA18 AGGTGACCGT 60 32

OPA19 CAAACGTCGG 60 35

OPC08 TGGACCGGTG 70 41

OPC12 TGTCATCCCC 60 39

OPC13 AAGCCTCGTC 60 39

OPC14 TGCGTGCTTG 60 38

OPC16 CACACTCCAG 60 38

OPC19 GTTGCCAGCC 70 38

OPC20 ACTTCGCCAC 60 38

OPS12 CTGGGTGAGT 60 38

OPAV03 TCACCGTGTC 60 38

OPAV06 CCCGAGATCC 60 38

4 Indonesian Journal of Agricultural Science Vol. 20 No. 1 June 2019: 01–08

of each local accession to environment, these are germplasms useful to develop new variety. Compared to the hybrids, the local papaya generally has a bigger fruit size which is more than 3,000 g in contrast to the hybrids which have the fruit size ranged from 400 to 2,500 g.

The two introduced accessions (Red Lady and Sunrise) were world well-known varieties. Red Lady is

the Mexican papaya having medium size and subtler flavor, while Sunrise or the strawberry papaya is the Hawaiian which has a smaller and sweeter fruit than the counterpart (Protain et al. 2010).

Papaya characters are highly influenced by environment. Light quality directly affects photosynthetic rates, whereas low quality of light

Table 3 . Fruit morphological characteristics of 22 papaya accessions.

Accessions Origin /parents Fruit shape Fruit size Flesh color Flesh texture

Flavor

BTK3 IAARD Pear shape √√√ Yellow Crunch +++BT2 IAARD Globule Reddish Soft ++ (with sour taste)BT3 IAARD Globule √ Reddish Soft ++

BT4 IAARD Globule √ Reddish Soft ++1XD BT1 X Dampit Elongate √√√ Orange - reddish Crunch ++

2X1 BT2 X BT1 Elongate √ Orange - reddish Soft +++G10 BT2 X Dampit Elongate √√√√ Orange - reddish Soft ++G15 BT3 X BTK Elongate √ Orange - reddish Crunch ++G18 Scc X D Club √√√√√ Orange Soft +Merah Delima IAARD Cylindrical √√√ Orange -– reddish Crunch +++Solinda IAARD Cylindrical √√ Yellow Crunch ++Carisya IPB Pear shape √ Reddish Crunch ++Cariso IPB Globule √ Orange Soft ++G28 West Sumatra, Indonesia Elongate √√√√√ Orange Soft +++

G30 West Sumatra, Indonesia Club √√√√√√ Orange Soft +++Dampit East Java, Indonesia Elongate √√√√ Orange- reddish Crunch ++Daun Jarak West Sumatra, Indonesia Elongate √√√√√ Orange Crunch +Tangkai Ungu West Sumatra, Indonesia Elongate √√√√√ Orange Crunch +Sicincin Panjang West Sumatra, Indonesia Club √√√√√√ Orange Crunch +Lokal Sumani West Sumatra, Indonesia Club √√√√√ Orange Crunch +Carica (outgroup) Yogyakarta, Indonesia Acron √ Yellow Crunch ++ (with fragrant)Red Lady Mexico Cylindrice √√√ Orange - reddish Crunch ++Sunrise Hawaii Globule √ reddish Soft +++

IAARD = Indonesian Agency for Agricultural Research and Development, IPB = Bogor Agricultural University, Fruit size, √ = small (< 500 g), √√ = medium small (500-1,000 g), √√√ = medium (1,000-1,500 g), √√√√ = medium big (1,500-2,000 g), √√√√√ = big (2,000-2,500 g), √√√√√√ = very big (>2500 g); Taste, + = slightly sweet, ++ = sweet, +++ = very sweet.

Fig. 1. Morphological characters of several Indonesia’s papaya accesssions; (a) 2X1, (b) Merah Delima, (c) Sicincin Panjang, and (d) Tangkai Ungu.

(a) (b) (c) (d)

5Genetic variability of Indonesian papaya accessions … (Riry Prihatini et al.)

reduces leaf thickness, petiole length, leaf weight, and leaf lobules. In addition, water availability is strongly related to fruit yield since the water directly affects photosynthetic rates. Water deficits reduce photosynthetic rates allowing to induce low productivity. While soil composition significantly affects fruit yield (Campostrini and Glenn 2007). All of these environmental factors contributed to the varied characters of papaya used in our study.

Fruit characters were generally influenced by pollination since each gametophyte carries different genetic materials to be inherited to the offspring (Arias-Calderon et al. 2014). Our results showed that based on the fruit morphological characters, the 23 papaya accessions were not differentiated into distinctive group. In papaya, fruit size and shape are sex linked traits which are also highly influenced by variety and environmental factors (Blas et al. 2012). Another study reported that the morphological characters sometimes fail to discriminate plant species. Thus, to complete the grouping, molecular analysis of these papaya accessions was necessary.

RAPD Polymorphism

The RAPD was chosen to assess the papaya genetic diversity due to its simplicity to produce accurate result without knowing targeted fragment or region in the genome. Among the 30 RAPD primers tested, 14 primers produced clear, strong, and reproducible bands, thus these markers were chosen for further analysis of 23 accessions. Some representative gel electrophoresis of papaya

accessions produced by RAPD primers were presented in Figure 2.

The 14 markers produced 91 bands, including 61 polymorphic bands (75.6%). Each primer amplified 4-9 bands with an average of 6.5 bands per primer and the band sizes of 100-800 bp. The detail of the amplified bands are summarized in Table 4.

Our study proved that the 14 RAPD primers used could resolve the genetic diversity of papaya accessions. These primers had the PIC values of 0.27 (OPY15) to 0.49 (RAPD2, RAPD3, and OPN09). The average heterozygosity (Hav) value, the effectiveness multiplex ratio (EMR), and the marker index (MI) were 0.40, 0.67, and 0.27, respectively. The PIC value is determined by both number and frequency of alleles. Since RAPD is a dominant marker, the highest PIC value for RAPD is 0.5 which assumes that there are two alleles per locus. Therefore, the PIC value for each marker in this study implied that the chosen markers were sufficient to examine the genetic diversity of papaya. As a comparison, PIC values in our study were correlated favorably with those on Jathropa (Ganesh Ram et al. 2008) and were higher than the PIC (0.33) observed on newly improved Indian papaya (Saran et al. 2015). Interestingly, the EMR and MI values of RAPD markers were lower than that of other markers, such as ISSR and SSR (Ismail et al. 2016). Thus in our study further analysis is applied to determine appropriately the genetic diversity and relationship among the papaya accessions.

Fig. 2. Example of RAPD-PCR products of 23 papaya accessions visualized on the 2% Agarose electrophoresis (M = 1 kb DNA ladder; 1 = Solinda, 2 = Merah Delima, 3 = Daun Jarak, 4 = Carisya, 5 = Dampit, 6 = Tangkai Ungu, 7 = BTK3, 8 = BT2, 9 = BT3, 10 = Sicincin Panjang, 11 = 2X1, 12 = 1XD, 13 = Lokal Sumani, 14 = G18, 15 = G30, 16 = G28, 17 = Red Lady, 18 = Sunrise, 19 = Cariso, 20 = BT4, 21 = G10, 22 = G15, 23 = Carica).

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

OPA02

RAPD3

OPY15

100 bp

400 bp

800 bp

100 bp

400 bp

800 bp

100bp

400 bp

800 bp

6 Indonesian Journal of Agricultural Science Vol. 20 No. 1 June 2019: 01–08

Genetic Diversity Analysis Based on RAPD

The Dice-Sorensen similarity test revealed that the farthest related accessions (0.71) were Sicincin Panjang and Carica, whereas the closest ones were Solinda, BTK3, G28, and G30 (0.98). The average similarity value was calculated as 0.85, indicating their narrow genetic diversity. It is in a good agreement with previous study on papaya germplasm using 11 RAPD markers that 10 domesticated Hawaiian papaya cultivars had a minimum similarity coefficient of 0.7, suggesting its narrow genetic diversity (Stiles et al. 1993). While 10 commercially Indian papaya cultivars were also detected to be narrow diversity using 18 RAPD markers (Sangeeta et al. 2005). However, 24 accessions of newly improved Indian papaya revealed a moderate diversity with similarity values of 0.30–0.99 according to 15 RAPD markers (Saran et al. 2015). Another report on 20 Indonesian papaya accessions using six RAPD primers showed their similarity coefficient of 0.00–1.00, indicating their low genetic diversity (Galingging 2005). These studies demonstrated that the number of RAPD markers probably did not influence the range of genetic diversity, but rather the choice of accession to be observed. The use of relatively similar genetic background of accessions would produce narrow genetic range, and vice versa.

The dendrogram constructed by the UPGMA is presented in Figure 3 and the three-D plot of the PCA based on the Eigen vector is shown in Figure 4. Based on 0.87 coefficient similarity, the Indonesian papaya accessions can be clustered into six different groups, including four groups with single member. Solinda and BTK3 which have yellow flesh color were grouped in the same cluster, indicating flesh color to be the morphological marker. Although there was no significant

correlation between morphological and genetic characters on papaya (Galingging 2005; Ocampo et al. 2006), flesh color on this study appeared to be one of the morphological characters which was substantiated by the RAPD marker as reported by Saran et al. 2015.

Similar with the genetic diversity index for total accessions, the calculated index for the hybrids was 0.34 and that for the local accessions was 0.36. This estimated gene diversity index in our study indicated that breeding could decrease genetic diversity due to repeated crosses. The clustering in Solinda and BT3 also implied their close similarity according to genetic characters rather than ecogeographical factors. Similar close distance occurred in BT2 and BT3, G28 and G30, as the consequence of their propagation method using seeds which produced similar genetic characters. Thus, small chances of cross breeding could be existed in the papaya propagation through seeds.

An interesting differentiation of a number of accessions appeared when assessed with RAPD polymorphism. Sicincin Panjang, Lokal Sumani, Cariso, and Carica were grouped as a single member cluster which represented their distinctive morphological characters. Sicincin Panjang is a local variety that has a very big and long fruit, originated from Padang Pariaman, West Sumatra. Sumani is a big fruit local variety from Solok, West Sumatra, and Carica is a local variety originated from Dieng Mountain of Central Java, far from the former two accessions. The Carica (Vasconcellea spp.) is an agro-economic highland papaya which was despite the similar morphology, was not the closest relative of Carica (d’Eeckenbrugge et al. 2014). The local varieties tend to self-breeding, thus resulted

Table 4. Data of amplified bands observed on 23 papaya accessions based on selected 14 RAPD primers.

Primer SequenceTotal number of

bands% of polymorphism

Unique bands

Range of fragment size (bp)

PIC value

RAPD1 GCGGGTGGAA 6 33,33 0 100-200 0.35

RAPD2 GTT TCGCTCC 4 100,00 0 100-400 0.49

RAPD3 GTAGACCC T 6 100,00 0 100-500 0.49

RAPD4 AAGAGCCCGT 7 57,14 0 100-600 0.46

RAPD5 AACGCGCAAC 8 25,00 0 100-500 0.31

RAPD6 CCCGTCAGCA 8 75,00 0 100-500 0.47

OPA01 CAGGCCCTTC 8 37,50 0 100-600 0.43

OPA02 TGCCGAGCTG 9 88,89 0 100-800 0.16

OPA11 CAATCGCCGT 5 80,00 0 100-600 0.38

OPA14 TCTGTGCTGG 5 60,00 1 150-400 0.49

OPC01 TTCGAGCCAG 7 71,43 4 100-700 0.44

OPC04 CCGCATCTAC 4 75,00 0 100-500 0.44

OPN09 TGCCGGCTTG 7 71,43 0 100-700 0.49

OPY15 AGTCGCCCTT 7 85,71 0 200-700 0.27

Hav: 0.40 EMR: 0.67 MI: 0.27

7Genetic variability of Indonesian papaya accessions … (Riry Prihatini et al.)

0.70 0.77 0.85 0.93 1.00

Dice-Sorensen coefficient of similarity

Solinda

BTK3

Merah Delima

Daun Jarak

Dampit

Tangkai Ungu I

1XD

BT2

BT3

2X1

Carisya

Sunrise

G18

G30

G28

Red Lady II

BT4

G10

G15

Sicincin Panjang III

Cariso IV

Lokal Sumani V

Carica VI

Red LadyG05

BT4

Lokal Sumani (V)

Cariso (V)0.28

Sicincin Panjang (III)Solinda

CarisyaBTK 3

Daun Jarak

2X11XD

Dampit

Merah Delima

Tangkai Ungu

Carica (VI)

Sunrise

III

G100.13

G18

G30G28

0.02

-0.170.29

0.160.03

-0.10-0.33

Y

-0.24 -0.12 -0.01 0.11 0.22X

BT3

Fig. 3. A dendogram shows genetic relationship among the 23 papaya accessions based on analysis of Dice-Sorensen coefficient of similarity and UPGMA.

Fig. 4. Three dimensional plot of 23 papaya accessions based on Eigenvector analysis based on RAPD polymorphism.

offspring having more homozygous alleles and unique characters (Rodríguez-Burruezo et al. 2008). The unique local papaya accessions in Indonesia should be preserved for further breeding program and genetic conservation. Two world well known accessions, i.e. Sunrise (Hawaiian accession) and Red Lady (Mexican accession) expectedly enhance to improve papaya varieties. Interestingly, the two accessions shared some molecular characteristics with the Indonesian hybrids and local accessions. Therefore, results of this study could be used as a reference to select appropriate parents for future breeding as well as to evaluate papaya hybrids.

CONCLUSION

Indonesian papaya genetic diversity was narrow according to RAPD polymorphism. A total of six groups were generated, of which the first and the second clusters consisted of mixed accessions, in contrast to the rest four clusters that each contained single accession of either local or hybrids. The higher genetic variation of 23 papaya accessions according to RAPD than that of morphological characters, indicated that molecular analysis could complement phenotypic information of papaya.

8 Indonesian Journal of Agricultural Science Vol. 20 No. 1 June 2019: 01–08

ACKNOWLEDGEMENT

We would like to acknowledge the Indonesian Agency for Agricultural Research and Development for funding this study.

REFERENCES

Arias-Calderon, R., Rouiss, H., de la rosa, R. & Leon, L. (2014) Variety and heretability of fruit characters in olive progenies from open-pollination. Scientia Horticulturae. 169, 94–98.

Asudi, G.O., Ombwara, F.K., Rimberia, F.K., Nyende, A.B., Ateka, E.M., Wamocho, L.S., Shitanda, D. & Onyango, A. (2010) Morphological diversity of Kenyan papaya germplasm. African Journal of Biotechnology. [Online] 9 (51), 8754–8762. Available from: doi:10.5897/ajb10.978.

Blas, A.L., Yu, Q., Veatch, O.J., Paull, R.E., Moore, P.H. & Ming, R. (2012) Genetic mapping of quantitative trait loci controlling fruit size and shape in papaya. Molecular Breeding. [Online] 29 (2), 457–466. Available from: doi:10.1007/s11032-011-9562-1.

Brown, J.E., Bauman, J.M., Lawrie, J.F., Rocha, O.J. & Moore, R.C. (2012) The structure of morphological and genetic diversity in natural populations of Carica papaya (Caricaceae) in Costa Rica. Biotropica. [Online] 44 (2), 179–188. Available from: doi:10.1111/j.1744-7429.2011.00779.x.

Campostrini, E. & Glenn, D.M. (2007) Ecophysiology of papaya: a review. Brazilian Journal of Plant Physiology. [Online] 19 (4), 413–424. Available from: doi:10.1590/S1677-04202007000400010.

Carvalho, F.A. & Renner, S.S. (2012) Molecular Phylogenetics and Evolution A dated phylogeny of the papaya family ( Caricaceae ) reveals the crop ’ s closest relatives and the family ’ s biogeographic history. Molecular Phylogenetics and Evolution. [Online] 65 (1), Elsevier Inc., 46–53. Available from: doi:10.1016/j.ympev.2012.05.019.

d’Eeckenbrugge, G.C., Drew, R., Kyndt, T. & Scheldeman, X. (2014) Vasconcellea for Papaya Improvement.In: Ming R., M.P. (ed.) Genetics and Genomics of Papaya. [Online] New York, NY, Springer New York, pp.47–79. Available from: doi:10.1007/978-1-4614-8087-7_4.

Dey, T., Saha, S. & Ghosh, P.D. (2015) Somaclonal variation among somatic embryo derived plants — Evaluation of agronomically important somaclones and detection of genetic changes by RAPD in Cymbopogon winterianus. South African Journal of Botany. [Online] 96, 112–121. Available from: doi:10.1016/J.SAJB.2014.10.010.

Doyle, J.J. & Doyle, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin. 19, 11–15.

Galingging, R.Y.B. (2005) Analisis keragaman 20 genotipe pepaya berdasarkan penanda morfologi dan RAPD (Random Amlified Polymorphic DNA). Sekolah Pascasarjana Institut Pertanian Bogor.

Ganesh Ram, S., Parthiban, K.T., Senthil Kumar, R., Thiruvengadam, V. & Paramathma, M. (2008) Genetic diversity among Jatropha species as revealed by RAPD markers. Genetic Resources and Crop Evolution. [Online] 55 (6), 803–809. Available from: doi:10.1007/s10722-007-9285-7.

Ismail, N.Z., Arsad, H., Samian, M.R., Ab Majid, A.H. & Hamdan, M.R. (2016) Evaluation of genetic diversity of Clinacanthus nutans (Acanthaceaea) using RAPD, ISSR and RAMP markers. Physiology and Molecular Biology of Plants. [Online] 22 (4), 523–534. Available from: doi:10.1007/s12298-016-0391-x.

Madarbokus, S. & Ranghoo-Sanmukhiya, V.M. (2012) Identification of genetic diversity among papaya varieties in Mauritius using morphological and molecular markers. International Journal of Live Sciences Biotechnology and Pharmacy Research. 1 (4), 153–163.

Nei, M. (1977) F-statistics and analysisof gene diversity in subdivided populations. Annals of Human Genetics. 41 (2), 225–233.

Nejatzadeh-Barandozi, F., Naghavi, M.R., Enferadi, S.T., Mousavi, A., Mostofi, Y. & Hassani, M.E. (2012) Genetic diversity of accessions of Iranian Aloe vera based on horticultural traits and RAPD markers. Industrial Crops and Products. [Online] 37 (1), 347–351. Available from: doi:10.1016/J.INDCROP.2011.12.013.

O. Aikpokpodion, P. (2012) Assessment of genetic diversity in horticultural and morphological traits among papaya (Carica papaya) accessions in Nigeria. Fruits. [Online] 67 (3), 173–187. Available from: doi:10.1051/fruits/2012011.

Ocampo, J., d’Eeckenbrugge, G.C., Buyere, S.B., de Bellaire, L.D.L. & Llitrault, P.O. (2006) Organization of morphological and genetic diversity of Carribean and Venezuelan papaya germplasm. Fruits. 61, 25–37.

Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J., Tingey, S. & Rafalski, A. (1996) 206 The comparison of RFLP, RAPD, AFLP and SSR markers for germplams analysis. Molecular Breeding. 2, 225–238.

Prihatini, R., Ihsan, F. & Indriyani, N.L.P. (2016) Genomic profiling of F1hybrids of durian (Durio zibethinus) revealed by RAPD-PCR. Journal of Horticultural Research. [Online] 24 (2), 69–76. Available from: doi:10.1515/johr-2016-0022.

Protain, S., Mohammed, M. & Wilson, L.A. (2010) Fruit ripening of Solo, Sunrise, Tainung 2, and Red Lady papaya at two temperatures. Journal of Applied Horticulture. 12 (1), 54–58.

Rodríguez-Burruezo, A., Prohens, J. & Nuez, F. (2008) Performance of hybrids between local varieties of Eggplant (Solanum melongena) and its relation to the mean of parents and to morphological and genetic distances among parents. European Journal of Horticultural Science. 73 (2), 1611–4426.

Rohlf, F.J. (2004) Numerical Taxonomy and Multivariate Analysis System. Setauket, NY, Exeter Publishing Ltd.

Roldán-Ruiz, I., Dendauw, J., Van Bockstaele, E., Depicker, A. & De Loose, M. (2000) AFLP markers reveal high polymorphic rates in ryegrasses (Lolium spp.). Molecular Breeding. [Online] 6 (2), 125–134. Available from: doi:10.1023/A:1009680614564.

Sangeeta, S., Ramesh, C., Anuj, P.S., Mishra, M., Pathak, R.K. & Ranade, S.A. (2005) Analysis of genetic diversity among papaya cultivars using Single Primer Amplification Reaction (SPAR) methods. The Journal of Horticultural Science and Biotechnology. [Online] 80 (3), 291–296. Available from: doi:10.1080/14620316.2005.11511932.

Saran, P.L., Choudhary, R., Solanki, I.S., Patil, P. & Kumar, S. (2015) Genetic variability and relationship studies in new Indian papaya (Carica papaya l.) germplasm using morphological and molecular markers. Turkish Journal of Agriculture and Forestry. [Online] 39 (2), 310–321. Available from: doi:10.3906/tar-1409-148.

Stiles, J.I., Lemme, C., Sondur, S., Morshidi, M.B. & Manshardt, R. (1993) Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars. Theoretical and Applied Genetics. [Online] 85 (6–7), 697–701. Available from: doi:10.1007/BF00225007.

Suketi, K., Poerwanto, R., Sujiprihati, S., Sobir & Widodo, W.D. (2010) Analisis kedekatan hubungan antar genotipe pepaya berdasarkan karakter morfologi dan buah. Jurnal Agronomi Indonesia. [Online] 38 (2) 130–137. Available from: doi:10.24831/JAI.V38I2.1797.

Vaidya, G. & Naik, G.R. (2014) Molecular identification of sex in Simarouba glauca by RAPD markers for crop improvement strategies. Biotechnology Reports. [Online] 4, Elsevier, 56–59. Available from: doi:10.1016/J.BTRE.2014.08.003.

Yap, I. V. & Nelson, R.J. (1996) A program for Performing Bootstrap Analysis of Binary Data to Determine the Confidence Limits of UPGMA-Based Dendrogram. Manila, International Rice Research Institute.