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http://journals.tubitak.gov.tr/agriculture/

Turkish Journal of Agriculture and Forestry Turk J Agric For(2015) 39: 310-321© TÜBİTAKdoi:10.3906/tar-1409-148

Genetic variability and relationship studies in new Indian papaya (Carica papaya L.) germplasm using morphological and molecular markers

Parmeshwar Lal SARAN1,*, Ravish CHOUDHARY1, Ishwar Singh SOLANKI1, Pravin PATIL2, Sanjay KUMAR2

1Indian Agricultural Research Institute Regional Station, Samastipur, Bihar, India 2National Bureau of Plant Genetic Resources, New Delhi, India

* Correspondence: plsdehradun@gmail.com

1. IntroductionPapaya (Carica papaya L.) is one of the most important fruits cultivated throughout the tropical and subtropical regions of the world. It is a native plant of tropical America and was introduced from the Philippines through Malaysia into India during the 16th century. Papaya germplasm shows considerable phenotypic variation for many horticultural traits (Ocampo et al., 2006). The different criteria that were used to estimate genetic diversity include pedigree records, morphological traits, and molecular markers (Weising et al., 2005). Plant taxonomy is traditionally dependent upon the comparative external morphological characters (Baxy, 2009). However, these markers are dependent on the environmental and the developmental stage. Therefore, molecular markers are the preferred choice for plant identification, as they are detectable in all tissues and are independent of environmental changes (Tapia et al., 2005).

Several molecular markers, including random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP), have been used for analysis of genetic diversity, relationships, and germplasm identification of papaya (de

Jesus et al., 2013). Among the molecular markers, RAPD and ISSR markers have been extensively used to study genetic diversity and relationships in papaya germplasm (Rodríguez et al., 2010; Sudha et al., 2013). Among these molecular markers, ISSRs are considered useful and have been extensively used for the identification of species or germplasms in a wide range of plants (Ahmad et al., 2010). ISSR amplifies intermicrosatellite sequences at multiple loci throughout the genome (Li and Xia, 2005) and permits the detection of polymorphisms in microsatellites and intermicrosatellite loci without previous knowledge of DNA sequences. These markers can detect polymorphisms in a single reaction with high repeatability and reproducibility. Knowledge of genetic variability is very useful for identifying the best combinations between germplasm lines with the potential to maximize the genetic gains attained by hybridization (Bertan et al., 2009).

The main objective of this study was to characterize the papaya germplasm lines using morphological and molecular markers in order to evaluate the genetic diversity and relationships among 24 germplasm lines, including new selections.

Abstract: Papaya germplasm showed wide morphological diversity in terms of fruit yield, weight, length, cavity, fruiting zone, flesh thickness, flesh color, and total soluble solids content. RAPD and ISSR markers also showed polymorphisms at rates of of 84.32% and 84.67%, respectively. Jaccard’s genetic similarity values of RAPD and ISSR were found in the range of 0.30–0.99 (average: 0.65) and 0.26–0.95 (average: 0.61), respectively, suggesting a moderate level of genetic diversity within the papaya group. Seven of the 15 RAPD primers and 5 of the 11 ISSR primers were also able to generate cultivar-specific amplicons, which were used for identification of germplasms Lalima, Madhu, PS-3, Farm Selection H, CO-7, and PSR-1-11. A dendrogram based on UPGMA separated all the selections into 4 main clusters, in which 3 genotypes, Lalima, Madhu, and PS-3, were found to be superior to the rest of the papaya germplasms. A two-dimensional plot generated from the principal component analysis of RAPD and ISSR data also supported the clustering pattern of the dendrogram. The present study revealed that morphological and molecular markers may be successfully utilized for determining genetic diversity and genetic relationships in papaya groups and may be used successfully in papaya breeding programs.

Key words: Papaya germplasm, genetic diversity, polymorphism, molecular markers, RAPD, ISSR

Received: 29.09.2014 Accepted: 01.12.2014 Published Online: 06.04.2015 Printed: 30.04.2015

Research Article

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2. Materials and methodsThe investigations were carried out for 2 consecutive years in 2012 and 2013 at the Indian Agricultural Research Institute Regional Station in Pusa, Samastipur, Bihar, India (52 m a.s.l.; 25.98°N, 85.67°E).2.1. Plant materialsPlants of 24 germplasm lines and their hybrids, representing native as well as foreign plants collected from different parts of India, were maintained and considered for the present study (Table 1). Newly emerged leaf samples of the cultivars were used for DNA extraction to avoid impurities like virus DNA, etc.

2.2. Morphological analysisTwenty-nine morphometric characters were evaluated using leaf, fruit, and seed specimens from 24 germplasm lines. Data on morphological characters were standardized using the YBAR option of the Stand program from the NTSYS-pc 2.1 software (Rohlf, 2000). Duplicate measurements for each specimen were averaged and were used to design a data matrix of pairwise similarities between germplasm lines. The simple matching coefficient (SMC) was used to measure the similarity, as it was the coefficient with the best results following a cophenetic test. Principal component analysis (PCA) was used to depict

Table 1. Collection details of papaya germplasms used for characterization.

Germplasm line State District Locality Latitude Longitude

Pusa Majesty Bihar Samastipur Pusa 25.98 85.68

Pusa Nanha Bihar Samastipur Pusa 25.98 85.68

Red Lady* Kolkata Barrackpore Anandapuri ------ ------

Pusa Delicious Bihar Samastipur Pusa 25.98 85.68

PSR2-8 Bihar Samastipur Pusa 25.98 85.68

Co-6 Tamil Nadu Coimbatore Pappanaicken Pudur 11.02 76.94

PS-2 Maharashtra Pune Sindhi Colony 18.55 73.81

Pusa Dwarf Bihar Samastipur Pusa 25.98 85.68

PSR Bihar Samastipur Pusa 25.98 85.68

Co-7 Tamil Nadu Coimbatore Pappanaicken Pudur 11.02 76.94

PSR-1-11 Bihar Samastipur Pusa 25.98 85.68

PS-1 Maharashtra Pune Sindhi Colony 18.55 73.81

PS-3 Maharashtra Pune Sindhi Colony 18.55 73.81

Mokama Local Bihar Begusarai Mokama 25.40 85.91

Farm Selection Uttarakhand Nainital Chakalua 29.14 79.21

Navya Bihar Samastipur Pusa 25.98 85.68

Madhu Bihar Samastipur Wasidpur 24.99 85.02

PL Gandak Bihar Samastipur Pusa 25.98 85.68

Vinayak** Gujarat Ahmedabad Ahmedabad ------ ------

Vinayak H** Gujarat Ahmedabad Ahmedabad ------ ------

PS-3 H Maharashtra Pune Sindhi Colony 18.55 73.81

CO-7 H Tamil Nadu Coimbatore Pappanaicken Pudur 11.02 76.94

Lalima Bihar Samastipur Pusa 25.98 85.68

Farm Selection H Uttarakhand Nainital Chakalua 29.14 79.21

*Purchased from East West Private Ltd., India.**Purchased from Vinayak Seeds, India.

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nonhierarchical relationships among the germplasm. Eigenvalues and eigenvectors were calculated by the Eigen program using a correlation matrix as input (calculated using standardized morphological data), and a 2-D plot was used to generate the two-dimensional PCA plot from NTSYS-pc 2.1 (Rohlf, 2000).2.3. Genomic DNA extraction and quantificationTotal genomic DNA was isolated from 24 germplasms using a cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle and Doyle, 1990) and was then quantified spectrophotometrically on an ND 1000 UV-spectrophotometer (Thermo Fisher Scientific Pvt. Ltd.).2.3.1. RAPD-PCR amplificationFifty decamer primers (Operon Technologies Inc.) were screened in the papaya germplasm, of which 15 primers generated polymorphic and reproducible banding patterns and were selected for final analysis. PCR amplification was carried out in a 25-µL reaction volume containing 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.0–2.5 mM MgCl2, 0.2 mM of each dNTP, 1.0 U of Taq DNA polymerase (Bangalore Genie), 10 pM primer, and 25–30 ng genomic DNA. The amplification was performed in a PTC-200 Thermocycler (MJ Research), with reaction conditions programmed as initial predenaturation at 94 °C for 4 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 37 °C for 1 min, and extension at 72 °C for 2 min. A final extension was done for 7 min at 72 °C. Amplification products were separated by electrophoresis on 1.5% agarose gels stained with ethidium bromide at 100 V for 3 h, and the bands were visualized and documented in AlphaImager HP (Alpha Innotech Corporation).2.3.2. ISSR-PCR amplificationA total of 100 primers identified by the University of British Columbia (UBC) were procured from Geno Biosciences Pvt. Ltd. and were used for ISSR-PCR optimization trials. Eleven primers, which gave the best amplification results with the sample DNA, were selected for final ISSR-PCR analysis. PCR amplification was carried out in a 25-µL reaction volume containing 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.0–2.5 mM MgCl2, 0.2 mM of each dNTP, 1.0 U of Taq DNA polymerase (Bangalore Genie), 0.2 µM primer, and 25–30 ng of genomic DNA. The amplification was performed in a PTC-200 thermocycler (MJ Research), with reaction conditions programmed as initial predenaturation at 94 °C for 4 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 2 min. A final extension was done for 7 min at 72 °C. Amplification products were separated by electrophoresis on 1.5% agarose gel stained with ethidium bromide at 100 V for 3 h, and the bands were visualized and documented in AlphaImager HP (Alpha Innotech Corporation).

2.4. Data analysisAmplified bands generated from RAPD and ISSR-PCR amplification were scored based on the presence (1) or absence (0) of bands for each primer and were used to calculate a genetic similarity matrix employing the SMC using NTSYS-pc version 2.1 (Rohlf, 2000). Cluster analysis was performed on both morphological and molecular data using the unweighted pair group method using arithmetic means (UPGMA) algorithm, from which dendrograms depicting the similarity among varieties were drawn and plotted using NTSYS-pc. The cophenetic correlation was calculated to find the degree of association between the original similarity matrix and the tree matrix in both morphological and molecular analyses. Using the Mantel test (Mantel, 1967), a comparison between both methods was performed for the accessions for which both data sets were available by calculating the correlation between the two data sets in NTSYS-pc. Using the same software, PCA was also carried out to identify any genetic association among the germplasms.

3. Results3.1. Morphological analysisThe papaya tree is small- to medium-sized. Fruit shape varies from ovate, round, or oblong to spheroid with a rounded, beaked, or truncated base and apex. Most of the fruits have a smooth surface texture. Flesh also varies between yellow, orange, and pink in color (Figure 1). The highest fruit yield/plant was observed in PS-3 (63.7 kg), followed by PSR (61.1 kg) and Navya (59.8 kg), whereas the lowest was found in Farm Selection H (5.4 kg), followed by PS-3 H (18.6 kg) and CO-7 (19.5 kg). The highest fruiting zone was observed in PS-3 (136.8 cm), followed by PS-3 H (134.8 cm) and PSR (119.6 cm), whereas the lowest was found in Pusa Dwarf, followed by Mokama Local (51 cm) and Pusa Nanha (51.6 cm). The highest fruit weight was 2.0 kg in PS-2, followed by 1.7 kg in PS-1 and 1.7 kg in PS-3, whereas the lowest was 0.50 kg in Farm Selection H, followed by 0.9 kg each in Pusa Majesty and Pusa Nanha. Flesh thickness was the highest in PS-2 (3.7 cm), followed by PS-1 (3.6 cm) and PS-3 (3.3 cm), whereas the lowest was observed in Pusa Majesty (2.5 cm), followed by Pusa Delicious (2.7 cm) and Vinayak (2.7 cm). The highest total soluble solid (TSS) content was obtained in Madhu (15.0 °Bx), followed by PL Gandak (14.0 °Bx) and Lalima (13.5 °Bx), whereas the lowest was observed in PSR 1-11 (7.4 °Bx), followed by PSR 2-8 (7.5 °Bx) and Mokama Local (7.9 °Bx) (Table 2).

Comparative analysis of 29 morphological characters revealed moderate variation. Pairwise similarity among the germplasm of C. papaya ranged from 0.04 to 0.89 with an average of 0.47 based on morphometric data. A dendrogram generated from morphometric data grouped

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all 24 germplasms into 4 major clusters (Figure 2a). The first cluster was the biggest, comprising 9 germplasm lines, and was subdivided in I-A and I-B. Subcluster I-A comprised 6 germplasm lines: Pusa Dwarf, Pusa Delicious, Farm Selection, Farm Selection H, Vinayak, and Vinayak H. Within this subcluster, the germplasm lines Vinayak and Vinayak H were quite similar morphologically, showing a similarity value of 0.89. Subcluster I-B comprised 3 germplasm lines: Pusa Nanha, Pusa Majesty, and PS-1. In this subcluster, Pusa Nanha was distinct from other germplasm lines with similarity value of 0.12. The second cluster comprised 8 germplasm lines: Navya, Lalima, Madhu, PL Gandak, PS-3, PS-3 H, Red Lady, and PS-2, in which PS-2 was distinct from rest of the germplasm lines and separated with the similarity value of 0.08. The third cluster comprised 4 germplasm lines, namely PSR, PSR-2-8, PSR-1-11, and Mokama Local, which was distinct from the rest of the germplasm lines with a similarity value of 0.09. The fourth cluster was the smallest one comprising 3 germplasm lines, namely CO-6, CO-7, and CO-7 H.

Based on Mantel Z-statistics (Mantel, 1967), the correlation coefficient (r) was estimated as 0.81. The r value of 0.81 was considered a good fit of the UPGMA cluster pattern to the data. The two-dimensional plot generated from PCA showed 3 groups that were found to be similar to the clustering pattern of the UPGMA dendrogram. In the 2-D plot, germplasm lines Vinayak, Vinayak H, PS-3, PS-3 H, and PS-2 constituted one separate group, whereas in UPGMA clustering, Vinayak and Vinayak H were grouped together in one cluster and PS-3, PS-3 H, and PS-2 were grouped in another cluster (Figure 2b). The analysis gave 18 principal components (PCs), out of which the first 10 principal components contributed 98.79% of the total variability. The first 5 principal components accounted for 87.17% of the total variability, and the first 3 accounted for 72.10% of the variance, in which the highest variation was contributed by the first component (31.74%), followed by second (23.80%) and third components (16.56%). The first PC was influenced by the characteristics of the fruit’s morphology, namely plant height, number of fruits/plant,

A

B

F E

H G I D

C

Figure 1. Morphological variation in papaya germplasm: fruiting zone (A) maximum and (B) Minimum; fruit yield/plant (C) maximum and (D) minimum; fruit size (E) maximum and (F) minimum; and flesh color: (G) orange, (H) yellow, and (I) pink.

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stem girth, number of leaves/plant, leaf width, leaf petiole length, fruit weight, fruiting zone, fruit yield/plant, fruit cavity width, and TSS (Table 3). In the second PC, the traits contributing to the total variability were number of nodes at first fruiting, leaf petiole length, flower length, bud drop, and mosaic (%). The third PC was mostly influenced by stem girth, leaf length, number of lobes/leaves, fruit length, diameter, texture and cavity length, flesh thickness and color, flower diameter, collar rot (%), bumpiness (%), leaf curl, and papaya ringspot virus (PRSV) severity (%).

3.2. RAPD analysisFifteen primers were selected for RAPD analysis based on reproducibility and banding patterns. A total of 185 bands were generated from 15 RAPD primers, of which 156 bands were polymorphic (84.32%) with an average of 10.4 polymorphic bands per primer. The fragment size ranged from 0.2 to 2.2 kb (Table 4). A representative gel profile was generated using primer OPL-08 (Figure 3a). Each primer amplified at a range of 6–19 amplicons with an average of 12.33 amplicons per primer. The OPL-

Table 2. Morphological traits of different Carica papaya germplasms.

Germplasm line Fruit yield/plant (kg)

Fruit weight(kg)

Fruiting zone (cm)

Flesh thickness (cm) TSS (°Br) Flesh

color

Pusa Majesty 33.10 0.90 76.80 2.50 10.20 Yellow

Pusa Nanha 31.30 0.90 51.60 3.00 9.00 Yellow

Red Lady 21.10 1.20 54.80 2.80 13.60 Pink

Pusa Delicious 36.30 1.20 73.40 2.70 10.30 Yellow

PSR 2-8 40.00 1.40 108.00 3.00 7.50 Yellow

Co-6 40.00 1.20 75.00 3.10 8.90 Orange

PS-2 49.20 2.00 98.60 3.70 12.90 Pink

Pusa Dwarf 43.60 1.00 51.00 3.00 10.00 Yellow

PSR 61.10 1.50 119.60 2.80 9.30 Yellow

Co-7 42.90 1.40 79.80 3.00 9.40 Orange

PSR 1-11 37.20 1.30 110.00 3.30 7.40 Yellow

PS-1 53.40 1.70 67.20 3.60 10.20 Yellow

PS-3 63.70 1.70 136.80 3.30 12.50 Pink

Mokama Local 42.20 1.60 51.00 3.10 7.90 Yellow

Farm Selection 46.10 0.90 65.20 3.00 10.30 Yellow

Navya 59.80 1.10 114.00 3.20 12.20 Yellow

Madhu 55.70 1.20 106.60 3.00 15.20 Orange

PL Gandak 50.40 1.20 100.00 3.00 14.00 Orange

Vinayak 37.50 1.30 56.40 2.70 10.90 Yellow

Vinayak H 19.60 1.00 56.40 2.70 10.90 Yellow

PS-3 H 18.60 1.30 134.80 3.30 12.50 Pink

CO-7 H 19.50 1.20 79.80 3.00 9.40 Orange

Lalima 53.40 1.10 115.20 3.10 13.50 Pink

Farm Selection H 5.40 0.50 65.20 3.00 10.30 Yellow

C.D. (P = 0.05) 3.01 3.47 2.83 3.37 3.58

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01 primer amplified the highest number of amplicons, 19, with 100% polymorphism, whereas OPL-09 primer generated the lowest number of amplicons, 6, with 83.33% polymorphism. The RAPD primer OPL-08 resulted in 15 amplicons with a size range between 0.28 and 2.2 kb with PIC value 0.317, and 2 distinct bands were observed in PSR-1-11, which distinguished it from other selected germplasm lines. The polymorphism percentage ranged from 53.33% (primer OPW-16) to 100% (primers OPL-01

and OPL-04). The average rate of polymorphism across all the 24 germplasm lines was 84.32%. Five RAPD primers amplified 7 unique bands in specific papaya germplasms. These primers amplified a specific DNA fragment that distinguished one germplasm line from the others. Patterns of distribution of bands across all germplasms revealed that primer OPL-02 amplified a single fragment in CO-7 (1250 bp), while primers OPL-05 (2100 bp), OPL-08 (1600 bp and 2200 bp), OPL-10 (1400 bp and 2000 bp),

Dim-1

–0.89 –0.49 –0.08 0.32 0.72

Dim

-2

–0.96

–0.59

–0.21

0.17

0.54

Pusa Dwarf

Pusa Nanha PSR

Navya

Pusa Delicious

Lalima PL-Gandak

Farm

Sele

ctio

n H

Co-6

Mokam

a Local

Coe�icient 0.04 0.26 0.47 0.68 0.89

Pusa Dwarf Farm Selection Farm Selection H Pusa Delicious Vinayak Vinayak H Pusa Nanha Pusa Majesty PS-1 Navya Lalima Madhu PL Gandak PS-3 PS-3H Red Lady PS-2 PSR PSR2-8 PSR-1-11 Mokama Local Co-7 CO-7 H Co-6

I

II

III

I

II

III

IV *'

+'

Figure 2. Dendrogram generated from morphological traits of 24 genotypes of C. papaya using UPGMA (A) and 2-D plot (B) methods.

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and OPW-16 (350 bp) amplified a single fragment in PSR-1-11. A maximum similarity value of 0.99 was observed between germplasm lines Navya and Farm Selection, indicating that they were genetically quite similar, whereas CO-7 and PS-3 H showed the lowest similarity coefficient of 0.30. Average similarity value across all the germplasm lines was 0.65.

A dendrogram generated based on the UPGMA method grouped all of the 24 germplasm lines into 4 major clusters. The first cluster comprised 6 germplasm lines: Pusa Majesty, Pusa Delicious, Pusa Nanha, Pusa Dwarf, PSR 2-8, and Red Lady. Within this cluster, Pusa

Nanha and Pusa Dwarf showed 90% genetic similarity, while Red Lady was distinct from other germplasm lines in this cluster with a similarity coefficient of 0.78. The second cluster comprised 5 germplasm lines, namely PSR-1-11, Farm Selection, Navya, PL Gandak, and Madhu. Within this cluster, Farm Selection and Navya were closely related with a similarity value of 0.99. The third cluster comprised 6 germplasm lines: Vinayak, PS-3 H, CO-7 H, Lalima, Farm Selection H, and Vinayak H. In this cluster, PS-3 H and CO-7 H showed 96% genetic similarity, while Vinayak H was distinct from the rest of the germplasm with a similarity value of 0.88. The fourth cluster was the

Table 3. Eigenvectors of morphological variables explained by first 3 principal components.

S. no. Characters PC1 PC2 PC3

1 Plant height (cm) 0.04 –0.01 –0.05

2 No of fruits/plant 0.23 0.18 –0.07

3 Stem girth (cm) 0.06 0.04 0.06

4 No. of nodes at first fruiting –0.09 0.08 –0.28

5 No. of leaves/plant 0.13 0.01 0.11

6 Leaf length (cm) 0.08 –0.01 0.09

7 Leaf width (cm) 0.11 –0.03 0.08

8 No of lobes/leaves 0.00 0.04 0.05

9 Leaf petiole length (cm) 0.02 0.02 0.00

10 Fruit petiole length (cm) –0.01 –0.08 –0.07

11 Fruit length (cm) –0.05 –0.06 0.10

12 Fruit diameter (cm) –0.04 0.05 0.10

13 Fruit weight (kg) 0.06 0.02 0.05

14 Fruiting zone 0.16 –0.09 –0.17

15 Flesh thickness (cm) 0.09 –0.07 0.18

16 TSS (°Br) 0.15 0.01 0.00

17 Fruit cavity length (cm) –0.01 –0.08 0.10

18 Fruit cavity width (cm) 0.15 –0.02 0.12

19 Flower length (cm) 0.05 0.07 0.06

20 Flower diameter (cm) 0.01 0.01 0.09

21 Collar rot (%) –0.20 0.14 0.54

22 Bumpiness (%) –0.45 –0.73 –0.06

23 Leaf curl (%) 0.32 –0.19 1.32

24 Bud drop (%) –0.81 0.56 –0.31

25 Mosaic (%) –0.45 0.20 –0.03

26 PRSV severity (%) –0.11 0.05 0.09

27 Fruit yield/plant 0.25 0.08 –0.18

28 Flesh color 0.13 0.01 0.27

29 Fruit texture 0.06 0.04 0.07

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Table 4. Details of amplified bands generated in 24 selections of C. papaya based on 15 RAPD and 11 ISSR primers.

PrimerSequence (5’-3’)RAPD

Total no. of bands

PBa PPBb (%)Unique band(s)

Range of fragment size (bp)

PIC value

OPL-01 GGCATGACCT 19 19 100.00 0 300–2000 0.345

OPL-02 TGGGCGTCAA 14 13 92.86 1 300–1550 0.351

OPL-03 CCAGCAGCTT 11 10 90.90 0 300–1750 0.367

OPL-04 GACTGCACAC 12 12 100.00 0 250–1900 0.368

OPL-05 ACGCAGGCAC 10 08 80.00 1 400–2100 0.283

OPL-06 GAGGGAAGAG 11 10 90.90 0 200–1800 0.388

OPL-07 AGGCGGGAAC 15 13 86.67 0 200–1600 0.366

OPL-08 AGCAGGTGGA 15 14 93.33 2 280–2200 0.317

OPL-09 TGCGAGAGTC 06 05 83.33 0 325–1300 0.354

OPL-10 TGGGAGATGG 10 07 70.00 2 400–2000 0.179

OPL-11 ACGATGAGCC 11 09 81.82 0 400–2200 0.321

OPN-07 CAGCCCAGAG 13 09 69.23 0 300–1400 0.308

OPW-16 CAGCCTACCA 15 08 53.33 1 300–1100 0.228

OPW-19 CAAAGCGCTC 11 09 81.82 0 350–1350 0.345

OPY-08 AGGCAGAGCA 12 10 83.33 0 250–1200 0.357

    185 156 84.32 7  

ISSR

UBC-807 AGAGAGAGAGAGAGAGT 12 10 83.33 1 300–1100 0.389

UBC-810 GAGAGAGAGAGAGAGAT 11 11 100.00 1 425–1250 0.419

UBC-812 GAGAGAGAGAGAGAGAA 14 10 71.43 0 200–1200 0.309

UBC-825 ACACACACACACACACT 15 11 73.33 1 300–1100 0.252

UBC-836 AGAGAGAGAGAGAGAGYA 09 07 77.78 0 200–1050 0.275

UBC-840 GAGAGAGAGAGAGAGAYT 09 09 100.00 0 350–1200 0.384

UBC-841 GAGAGAGAGAGAGAGAYC 11 10 90.90 1 260–1300 0.387

UBC-855 ACACACACACACACACYT 10 10 100.00 0 370–1300 0.457

UBC-857 ACACACACACACACACYC 13 11 84.62 2 300–1500 0.302

UBC-858 TGTGTGTGTGTGTGTGRT 11 09 81.82 1 250–1200 0.344

UBC-864 ATGATGATGATGATGATG 05 03 60.00 0 200–1250 0.207

  120 101 84.67 7  

a Total polymorphic bands; b Percentage of polymorphic bands.

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largest one and consisted of 7 germplasm lines, i.e. CO-6, CO-7, PS-2, PS-3, PSR, PS-1, and Mokama Local. Within this cluster, CO-6 and CO-7 were closely related with a similarity value of 0.93, whereas PS-3 was distinct from rest of the germplasm lines, with a similarity value of 0.74.

Based on Mantel Z-statistics (Mantel, 1967), the correlation coefficient (r) was estimated to be 0.79. The 2-D plot generated from PCA of RAPD data was also in coherence with the clustering pattern of the UPGMA dendrogram. The exception was PSR-1-11, which was distinctly separated in the 2-D plot; in the dendrogram, it was grouped in cluster II. The first and second PCs accounted for 31.18% and 18.15%, respectively, of the total variation.3.3. ISSR analysisEleven primers were selected for ISSR analysis based on reproducibility and banding patterns. A total of 120 bands were generated, of which 101 bands were polymorphic (84.61%). Each primer amplified 3–11 polymorphic amplicons with an average of 9.18 amplicons per primer. The primers UBC-810, UBC-825, and UBC-857 amplified the highest number of amplicons at 11, whereas UBC-864 amplified the lowest number of polymorphic amplicons at 3. Figure 3b showed a representative ISSR profile generated by primer UBC-857. The polymorphism percentage ranged from 60% (primer UBC-864) to 100% (primers UBC-810 and UBC-840). Average polymorphism rate across all 24 germplasm lines was 80%. The overall size of the PCR-amplified fragments ranged from 200 bp to 1500 bp (Table 4). Patterns of distribution of bands across all accessions of C. papaya revealed that primer UBC-807 (300 bp) for Madhu, UBC-810 (1250 bp) for Red Lady, UBC-825 (350 bp) for CO-7, UBC-841 (1250 bp) for Farm Selection H, UBC-857 (1500 bp) for PS-3, (1300 bp) for Lalima (Figure 3b), and UBC-858 (1150 bp) for Farm Selection H amplified a unique DNA fragment that distinguished the respective cultivar from the others.

The pairwise Jaccard’s similarity coefficient among all of the 24 germplasm lines ranged from 0.26 to 0.95. The maximum similarity of 0.95 was observed between germplasm lines Lalima and PS-3 H, indicating that they are genetically quite similar, whereas Lalima and CO-7 showed the minimum similarity coefficient of 0.26. Average similarity across all the germplasm lines was 0.61. In the dendrogram, all of the 24 germplasm lines were grouped into 4 major clusters. The first cluster comprised 7 germplasm lines: Pusa Majesty, Pusa Delicious, PSR 2-8, Pusa Nanha, Pusa Dwarf, PSR 1-11, and Red Lady. In this cluster, Pusa Delicious and PSR 2-8 were the most closely related with 87% similarity, whereas Red Lady was diverse from the other germplasm lines of this cluster with a similarity value of 0.67. The second cluster comprised 4 germplasm lines: Farm Selection, Navya, PL Gandak, and

Madhu. Farm Selection and Navya showed 93% genetic similarity, whereas Madhu was most distinct from other germplasm lines with a similarity value of 0.82. The third cluster comprised 6 germplasm lines: Vinayak, CO-7 H, PS-3 H, Lalima, Vinayak H, and Farm Selection H. PS-3 H and Lalima showed the highest genetic similarity (95%). The fourth cluster comprised 7 germplasm lines, namely CO-6, PS-2, PS-1, PS-3, PSR, CO-7, and Mokama Local. Within this cluster, PS-2 and CO-6 were most closely related with a similarity value of 0.80, while PSR was distinct from the rest of the germplasm lines with a similarity value of 0.69.

Based on Mantel Z-statistics (Mantel, 1967), the correlation coefficient (r) was estimated to be 0.79. The 2-D plot generated from PCA of ISSR data was also consistent with the clustering pattern of the UPGMA dendrogram. The exceptions were PS-1 and PS-2, which were grouped with Red Lady in the 2-D plot but were grouped in cluster IV in the dendrogram. The first and second PCs accounted for 29.12% and 16.73%, respectively, of the total variation.3.4. Cumulative data analysis of morphology, RAPD and ISSRPairwise similarity among the germplasms ranged from 0.30 to 0.85 with an average of 0.58 based on combined morphometric, RAPD, and ISSR data. The highest similarity (85%) was observed between the CO-7 H and Vinayak germplasm lines, whereas the lowest was observed between CO-7 and Lalima with a similarity value of 0.30. A dendrogram based on combined morphometric, RAPD, and ISSR data grouped all 24 germplasm lines into 4 major clusters (Figure 4a). The first cluster comprised 5 germplasm lines, namely Pusa Majesty, Pusa Delicious, PSR 2-8, Pusa Nanha, and Red Lady. Within this cluster, Pusa Nanha and PSR 2-8 were the most similar morphologically and genetically, showing a similarity value of 0.77. In this group, Red Lady was distinct from the other germplasm lines, with a similarity value of 0.68. The second cluster comprised 7 germplasm lines: Pusa Dwarf, PSR 1-11, Farm Selection, Navya, PL Gandak, Madhu, and Lalima. Within this cluster, Farm Selection and PL Gandak were observed to be quite similar with a similarity value of 0.84. The third cluster comprised 4 germplasm lines: Vinayak, CO-7 H, Farm Selection H, and Vinayak H; Vinayak and CO-7 H showed the highest genetic similarity (85%). The fourth cluster was the biggest one, comprising 8 germplasm lines, namely CO-6, PS-2, CO-7, PSR, PS-1, Mokama Local, PS-3, and PS-3 H. Within this cluster, CO-6 and PS-2 were the most similar (0.76), whereas CO-7 was distinct from rest of the germplasm lines with a similarity value of 0.66. The 2-D plot generated from the PCA of the combined morphometric, RAPD, and ISSR data (Figure 4b) also supported the clustering pattern of the UPGMA dendrogram. In the 2-D plot, the genotype PS-3 H was

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grouped in cluster I, whereas in UPGMA clustering, it was grouped in cluster IV. The analysis gave 22 PCs, out of which the first 10 PCs contributed 81.13% of the total variability of the analyzed germplasm. The first 5 PCs accounted for 61.75% of the total variability; the first 3 accounted for 46.17% of the variance, in which maximum variability was contributed by the first component (22.31%), followed by the second (14.59%) and third (9.27%) components.

4. DiscussionMorphological, RAPD, and ISSR marker analyses among the papaya germplasm lines were successfully employed to estimate genetic variability and to establish genetic relationships. Morphological characterization based on 29 characters revealed significant diversity (62%) in leaf, fruit, and stem traits. The RAPD and ISSR data generated from the 24 germplasm lines with 15 and 11 primers, respectively, were sufficient to provide inferences on genetic differentiation and relationships. The RAPD and ISSR markers showed a high level of polymorphism (84.5%). However, a low level of polymorphism (29.2%) was reported by Sudha et al. (2013) and de Jesus et al.

(2013) in papaya based on ISSR markers. PIC values were also recorded high with both RAPD (0.325) and ISSR (0.339) markers, showing the efficiency of molecular markers used to detect polymorphism within the papaya group.

Jaccard’s genetic similarity values of RAPD and ISSR were found in the ranges of 0.30–0.99 (average: 0.65) and 0.26–0.95 (average: 0.61), respectively, suggesting a moderate level of genetic diversity within the papaya group. This moderate level of genetic diversity within the papaya group may be due to free genetic exchange within papaya cultivars as well as closely related species. In all the dendrograms (morphometric, RAPD, ISSR, and combined), Pusa Nanha, Pusa Delicious, and Pusa Majesty were grouped together in the same cluster, showing a close genetic relationship. These cultivars are the result of natural selection and chance seedlings derived from C. papaya. Navya and Madhu found a place in the same cluster in RAPD, ISSR, morphometric, and combined dendrograms, suggesting high genetic similarity between the cultivars. These are the natural selections of papaya having similar morphology and adapted to higher altitudes in particular

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@!Figure 3. Representative gel profiles of C. papaya based on RAPD and ISSR primers (A) OPL-08 and (B) UBC-857, respectively, showing unique bands (circled bands are distinguishing the respective germplasm line from other selected lines).

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climatic conditions of the Northeastern Plains Zone of India. The germplasm lines CO-6 and CO-7 were grouped together, as both of them originated from the south of India. Lalima and PS-3H were observed to be in same cluster in RAPD and ISSR dendrograms, suggesting high genetic similarity because Lalima may be a cross between PS-3 and Navya. The grouping of all 24 germplasms in

the dendrogram was congruent with the morphology and genetic origin of cultivars/germplasm lines.

Morphological characters like plant height, stem girth, leaf petiole length, number of leaves/plant, leaf length and width, number of fruits/plant, fruiting zone, fruit weight, cavity width, yield/plant, texture, flower length and diameter, leaf curl, flesh thickness and color, and TSS

Coe�icient 0.45 0.55 0.65 0.75 0.85

Pusa Majesty Pusa Delicious Pusa Nanha PSR2-8 Red Lady Pusa Dwarf PSR-1-11 Farm Selection PLGandak Navya Madhu Lalima Vinayak CO-7 H Farm Selection H Vinayak H Co-6 PS-2 Co-7 PSR PS-1 Mokama Local PS-3 PS-3 H

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Figure 4. UPGMA dendrogram (A) and 2-D plot (B) of 24 germplasm lines of C. papaya generated based on combined morphometric, RAPD, and ISSR data.

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representing the maximum variability as revealed by the first 3 principal components were identified for developing a minimal descriptor. Sudha et al. (2013) also reported morphological diversity in terms of fruit weight, length and girth, flesh thickness, and TSS. This is the first study where morphological markers have been substantiated in combination with RAPD and ISSR markers to distinguish papaya germplasms.

Both morphological and molecular markers showed a high degree of variation among the selected papaya germplasm lines. The present study revealed that morphological markers and both of the molecular markers can be successfully utilized for inferring genetic diversity and genetic relationships in papaya. Lalima, Madhu, and PS-3 are different germplasm lines owing to morphological

variation, but it was confirmed using molecular markers that the Lalima and Madhu genotypes are genetically close. Similarity among Pusa Dwarf, Pusa Nanha, Pusa Delicious, and Pusa Majesty further confirmed the importance of these markers for distinguishing the papaya cultivars. Overall, Lalima and Madhu were found to be superior new genotypes on the basis of fruit yield and quality. Results derived from this study would be highly useful in papaya breeding programs and may be used for further crop improvement using advance marker systems.

AcknowledgmentThe authors are grateful to the Director, NBPGR, and the Director, IARI, New Delhi, for providing the laboratory facilities and financial support.

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