An Analysis on DNA Fingerprints of Thirty Papaya Cultivars (Carica papaya L.), Grown in Thailand with the Use of Amplified Fragment Length Polymorphisms Technique
Janthasri Ratchadaporn ,
The experiment was carried out at the Department of Horticulture, Ubon Ratchathani University, Ubon Ratchathani province, Northeast Thailand during June 2002 to May 2003 aims to identify DNA fingerprints of thirty papaya cultivars with the use of Amplified Fragment Length Polymorphisms (AFLP) technique. Papaya cultivars were collected from six different research centers in Thailand. Papaya plants of each cultivar were grown under field conditions up to four months then leaf numbers 2 and 3 of each cultivar (counted from top) were chosen for DNA extraction and the samples were used for AFLP analysis. Out of 64 random primers being used, 55 pairs gave an increase in DNA bands but only 12 pairs of random primers were randomly chosen for the final analysis of the experiment. The results showed that AFLP markers gave Polymorphic Information Contents (PIC) of three ranges i.e., AFLP markers of 235 lied on a PIC range of 0.003-0.05, 47 for a PIC range of 0.15-0.20 and 12 for a PIC range of 0.35-0.40. The results on dendrogram cluster analysis revealed that the thirty papaya cultivars were classified into six groups i.e., (1) Kaeg Dum and Malador (2) Kaeg Nuan (3) Pakchong and Solo (4) Taiwan (5) Co Coa Hai Nan and (6) Sitong. Nevertheless, in spite of the six papaya groups all papaya cultivars were genetically related to each other where diversity among the cultivars was not significantly found.
to cite this article:
Janthasri Ratchadaporn , Katengam Sureeporn and U. Khumcha , 2007. An Analysis on DNA Fingerprints of Thirty Papaya Cultivars (Carica papaya L.), Grown in Thailand with the Use of Amplified Fragment Length Polymorphisms Technique. Pakistan Journal of Biological Sciences, 10: 3072-3078.
Papaya (Carica papaya L.), one of many important cash crops is believed
to have its origin from Central America i.e., Mexico, Costa Rica and later spread
to the nearby countries during the fifteenth century and soon this crop was
introduced to West Indies and arrived in the Philippines in the sixteenth century
and later to Indonesia and the nearby countries such as India, Thailand and
others. This crop reached African countries in the eighteenth century (1874).
Papaya is, more or less, a tropical crop and the plants can thrive on easily
in most soils. Papaya cultivation can be found in many countries such as Australia,
India, Burma, USA, Thailand, Taiwan and some other countries in Africa and also
in most Latin American countries and many other countries in the tropics (Suksri,
1999). Papaya fruits provide nutritious value to man, particularly when ripe.
It is rich in vitamins C and A, whilst green fruits contain an enzyme known
as papain or proteolytic enzyme, which could be used to tenderise or soften
animal meats when cooking, particularly meats of beef or pork and it is commercially
used as a meat tenderiser and it is also used in many other chemical products
(Williams, 1975; Purseglove, 1968; Smith et al., 1992). A large amount
of papain has been imported to Thailand annually for industrial uses (Waraporn,
1990). In the 2002, Thailand used an area of approximately 29,048 hectares for
papaya plantations with its annual production of fruits of approximately 467,983
metric tones. The popular varieties being cultivated in Thailand include Kaeg
Dum, Kaeg Nuan and Co Coa, whilst other varieties such as Florida and Hawaiian
received less attention from growers. Papaya plantations in Northeast Thailand
occupied the largest land area of 57% followed by the Central Plane region,
whilst growers in other regions grow papaya mostly for their household consumption
(Anonymous, 2001 and 2002). The local population in each province of the country
weekly consumed several tones of fresh papaya fruits apart from ripen fruits
where they use papaya fresh fruits for papaya salad mostly in northeastern region.
Papaya salad is normally prepared for a daily diet of the local population in
Northeast Thailand and it is known as the fabulous Som Tam. It is a part of
daily life of the people thus this crop signifies its tangible economic impact
of the country.
For the past few decades there had been some changes in using different types of breeds due to the changing in environmental conditions and selection processes and the use of natural hybridization, thus growers were not able to distinguish vividly, which papaya plants could retain its true to type of its origins, hence growers had given their own appropriate names for their selected offsprings of cultivars. It has been advocated that AFLP technique could provide outstanding results since it could differentiate DNA of different living organisms with a high degree of accuracy (Cerrera et al., 1996) and the technique had been used by several workers to identify some certain orchard plant species such as in citrus (Ulubelde and Tan, 1986), mangoes (Degani et al., 1995), longan (Ratchadaporn, 2003) and others. It seems more likely that information on genetic code identification of papaya plants in Thailand is limited due to presumably the complication in carrying out the work or perhaps the lack of equipment. Therefore, it may be of tangible value to investigate and identify different types of popular papaya cultivars being cultivated within the country with the use of a technique known as AFLP. The technique includes the use of Polymerase Chain Reaction (PCR) to determine genomic DNA where it provides fingerprints of its actual DNAs of their genetic codes without the interference of environmental conditions (Sharma et al., 1996).
MATERIALS AND METHODS
This study was carried out at the Department of Horticulture, Faculty of Agriculture,
Ubon Ratchatani University, Ubon Ratchatani province, Northeast Thailand during
June 2002 to May 2003 to search for more information on popular papaya cultivars
being cultivated in Thailand, particularly in northeastern region where a large
amount of papaya fruits has been consumed annually. The search includes the
collection of seeds of different types and local names of papaya cultivars from
six different places namely: (1) Srisaket Horticultural Research Centre. There
were twenty cultivars obtained from this research center, they include Tha Phra
1, Tha phra 2, Tha Phra 3, Khaeg Dum Dum Nern, Sitong, Khaeg Dum Dum Nern x
Malador, Pak Chong, Khaeg Dum Dum Nern x Pak Chong, Khaeg Dum Srisaket-8-9,
Solo, Co Coa Karn Dum, Khaeg Dum Srisaket-6-2, Khaeg Dum Srisaket, Khaeg Dum
Nakorn Phanom, Mexico-Indonesia, Co Coa Klong Tor, Co Coa Som Dang, Malador,
Taiwan x Khaeg Dum Dum Nern and Khaeg Dum Dum Nern x Mexico-Indonesia; (2) Loei
Highland Agricultural Station, this location provided five papaya cultivars,
they include Khaeg Dum Trat, Khaeg Dum Loei, Hai Nan, Taiwan and Taiwan Tissue;
(3) Phichit Horticultural Research Centre, only one cultivar was attained, i.e.,
Khaeg Nuan; (4) Department of Horticulture, Kaset Sart University where Khaeg
Nuan x Malador cultivar was obtained; (5) Khon Kaen Horticultural Research Centre
with one cultivar i.e., Khaeg Dum Khon Kaen and (6) Department of Horticulture,
Ubon Ratchathani University with one cultivar of Khaeg Dum Ubon Ratchathani.
Some considerable amounts of matured seeds of 30 different papaya cultivars
were obtained from these six locations and most of them had reached its generations
of F7 after several years of selection. Seeds of each cultivar were sown separately
into trays filled with moist compost and then placed the trays on germinating
desks under glasshouse conditions. Watering was carried out daily to assure
adequate amount of moisture content of the compost in each tray. Eight to fourteen
days after sowing, papaya seeds were germinated. One week after germination
healthy seedlings of each tray were transplanted into polythene pots, each pot
contained approximately 500 g of moist compost. Thirty polythene pots were used
for each cultivar and they were allowed to grow under glasshouse conditions
for 2 months after germination. Watering of seedlings was carried out daily
to assure no wilting signs of leaves found during this growing period. After
2 months of growing period under glasshouse conditions then 20 healthy seedlings
of each cultivar were chosen at random and transplanted into the Experimental
Field, Ubon Ratchatani University at a distance between rows and within rows
of 4x4 m, respectively. Each seedling was allowed to grow in a drill where an
amount of soil of 0.50 m3 was taken out to make a hole with a dimension
of 50x50x50 cm and then a similar quantity of topsoil was thoroughly mixed with
cattle manure of 2-kg (approximately 30% moisture contents) together with 200
g of a complete chemical fertiliser 15-15-15 (N, P2O5, K2O)
and then the mixture was used to replace the amount of soil taken out from each
hole of each papaya plant stand. Due to the fact that most soils in Northeast
Thailand are the soils of poor fertility as reported by Suksri (1999), Kasikranan
(2003) and Pholsen (2003), thus to attain normal growth and high yield of papaya
plants in each hole of papaya plant stand, growing media for papaya seedlings
must be thoroughly prepared in order to provide adequate amounts of soil nutrients
and aeration for respiration of roots of papaya plants. Leaf samples of papaya
plants for the determinations of DNA were taken from a 4-moth old papaya plants.
At 4 months after germination, all papaya plants started to bear a number of
fruits hence uniformed papaya plants (observed by naked eye) were chosen at
random for leaf samples. Papaya young leaves of numbers 2 and 3 (being counted
from top to lower leaves) were cut at the edge of petiole of leaves and then
collected for laboratory assessment. The bearing fruits of papaya plants facilitated
uniform characteristics of papaya plants to be chosen, thus leaf samples of
those bearing fruits were collected. Papaya leaf samples of the same cultivar
were collected from 15 papaya plants and then they were used for DNA extraction.
The extraction was carried out with the method of Aggarwal et al. (1999)
where two types of enzymes were used i.e., EcoRI and MSel. The extracted DNA
samples were mixed together according to their respective cultivars and then
the samples were used for Amplified Fragment Length Polymorphisms (AFLP). The
AFLP analysis was carried out with the use of the method of Vos et al. (1995)
where 64 pairs of AFLP random primers were used and 55 pairs of random primers
were able to produce DNA bands. However, with this study only 12 pairs of AFLP
random primers were randomly chosen. They include E-AGG/M-CTC; E-AGG/M-CAT;
E-AGC/M-CAA; E-ACT/M-CTG; E-ACT/M-CAG; E-ACG/M-CTC; E-ACC/M-CTG; E-ACC/M-CAT;
E-ACA/M-CAA; E-AAG/M-CAC; E-AAC/M-CTA and E-AAC/M-CAC. The attained results
were statistically calculated with the use of the computer programmes where
the calculation started with the use of Numerical Taxonomy and Multivariate
Analysis Systems (NTSYS, pc2.02i) of Rohlf (1998) to produce Nei and Li similarity
correlation coefficients (Nei and Li, 1979) and then matrix genetic distances
were used for cluster analysis of genotypic values for a dendrogram figure with
the application of un-weighted pair group method on the basis of arithmetic
averages (UPGMA) as carried out by Sneath and Sokal (1973).
AFLP primers, monomorphic bands, polymorphic bands and polymorphic %: With
the use of 64 pairs of primers being applied to 30 papaya cultivars, the results
showed that there were 9 pairs of primers where an increase in DNA bands was
not found. However, there were 55 pairs of primers where an increase in DNA
bands was possible but for this study only 12 pairs of primers were randomly
chosen. They include (1) E-AGG/M-CTC, (2) E-AGG/M-CAT, (3) E-AGC/M-CAA, (4)
E-ACT/M-CTG, (5) E-ACT/M-CAG, (6) E-ACG/M-CTC, (7) E-ACC/M-CTG, (8) E-ACC/M-CAT,
(9) E-ACA/M-CAA, (10) E-AAG/M-CAC, (11) E-AAC/M-CTA and (12) E-AAC/M-CAC, these
12 pairs of primers were used for this investigation (Table 1).
||Pairs of AFLP primers being used, numbers of bands primer-1,
monomorphic bands primer-1, polymorphic markers primer-1
and % of polymorphic bands primer-1of 36 popular papaya cultivars,
grown in Thailand
The results showed that DNA bands primer-1 ranged from 50 to 75
for E-AGC/M-CAA and E-ACC/M-CAT, respectively with a total DNA bands or AFLP
markers of 734 bands with an average value of 63 bands. For monomorphic bands,
it ranged from 11 to 33 bands for E-AGC/M-CAA and E-ACG/M-CTC, respectively
and out of 734 bands it gave a genetic similarity of bands of 249. With the
results on polymorphic bands, the results showed that polymorphic bands ranged
from 32 to 43 bands for E-AAC/M-CAC and E-AGG/M-CTC, respectively with total
polymorphic markers of 485 bands, i.e., 66.08% of all DNA bands (734 bands).
Polymorphic information content (PIC): With PIC, the results showed that polymorphic markers of 485 bands of the 30 papaya cultivars gave a highest value of AFLP markers of 235 where it lied on a PIC range of 0.003-0.05 i.e., equivalent to 48.45% out of all DNA bands. It was shown that values of AFLP markers gave a range of values from 12 to 235 where the second lower value was 47 with a PIC range of 0.15-0.20 and the lowest value of 12 gave a PIC range of 0.35-0.40 (Fig. 1).
Cluster analysis on genetic evaluation: The results on cluster analysis
derived from a dendrogram (Fig. 2) revealed that papaya cultivars
being used in this study could be classified into six groups of papaya cultivars
i.e., Group I (Kaeg Dam and Malador group), this group includes 18 papaya cultivars
they are: Tha Phra 1, Tha Phra 2, Tha Phra 3, Kaeg Dum Khon Kaen, Kaeg Dam Kalasin,
Kaeg Dum Loei, Kaeg Dum Nakhon Phanom, Kaeg Dum Ubon, Kaeg Dum Trat, Kaeg Dum
Dum Nern x Pak Chong, Kaeg Dum Dam Nern x Mlador, Kaeg Dum Srisaket 4-9, Kaeg
Dum Srisaket 9-5, Kaeg Dum Srisaket, Kaeg Dum Dum Nern, Malador, Taiwan x Kaeg
Dum Dum Nern and Kaeg Dum Dum Nern x Mexico-Indonesia; Group II (Kaeg Nuan group),
this group consisted of 2 cultivars i.e., Kaeg Nuan and Kaeg Nuan x Malador;
Group III (Pakchong and Solo group), this group has 2 papaya cultivars i.e.,
Pak Chong and Solo; Group IV (Taiwan group), this group has 2 cultivars i.e.,
Taiwan and Taiwan Tissue; Group V (Co Coa Hai Nan, Mexico-Indonesia group),
this group consisted of 5 papaya cultivars i.e., Co Coa Karn Dam, Mexico-Indonesia,
Hai Nan, Co Coa Klong Tor and Co Coa Som Dang; Group VI (Sitong group), this
group has a single cultivar i.e., Sitong.
||The distribution of polymorphic information contents (PIC
coefficients) of 30 papaya cultivars being popularly grown in Thailand as
distributed by Amplified Fragment Length Polymorphisms (ALFP) markers
||A dendrogram structure illustrated similarity coefficients
and relationships among 30 papaya cultivars based on cluster analysis of
NTSYS-pc2.02i (Rohlf, 1998). P = Papaya cultivars. P1= Tha Phra1, P2 = Tha
Phra2, P3 = Tha Phra3, P4 = Kaeg Dum Khon Kaen, P5 = Kaeg Dum Dum Nern,
P6 = Kaeg Dum Trat, P7 = Kaeg Nuan, P8 = Sitong, P9 = Kaeg Dum Kalasin,
P10 = Kaeg Dum Dum Nern x Pak Chong, P11 = Pak Chong, P12 = Kaeg Dum Dum
Nern x Malador, P13 = Kaeg Dum Loei, P14 = Kaeg Dum Srisaket-8-9, P15 =
Kaeg Nuan x Malador, P16 = Solo, P17 = Co Coa Karn Dum, P18 = Kaeg Dum Srisaket-6-2,
P19 = Hai Nan, P20 = Taiwan, P21 = Taiwan Tissue, P22 = Kaeg Dum Srisaket,
P23 = Kaeg Dum Nakhon Phanom, P24 = Kaeg Dum Ubon, P25 = Mexico-Indo, P26
= Co Coa Klong Tor, P27 = Co Coa Som Dang, P28 = Malador, P29 = Taiwan x
Kaeg Dum Dum Nern and P30 = Kaeg Dum Dum Nern x Mexico-Indonesia
In Thailand, the plantation of papaya crop within a few decades was carried out mainly in the Central Plane region where growers planted a large number of papaya plants within a large piece of land such as those plantations carried out at Ratchaburi province and others. The plantations aimed to supply papaya fresh fruits mainly to northeastern region where tones of fresh fruits have been used mostly for papaya salad or by the local name of Som Tam. Within this decade many Thai government agencies had paid their attention to preserve and collect germ plasms of papaya cultivars in order to search for outstanding cultivars for growers in all regions of the country such as Srisaket Horticultural Research Center, Khon Kaen Horticultural Research Center and many other centers as stated earlier in this study. It is generally known to the Thai people that papaya is an important crop being grown for household consumption hence a large number of the families in each village grows a few plants of papaya for their own utilization. Thus growers normally preserve their own cultivars from generation to generation. It was found with the results carried out in Thailand by Sukhontip (2003) with the use of DNA amplification fingerprints (DAF) that from 11 papaya cultivars being used for the experiment, the papaya cultivars could be classified into three groups only i.e., (1) Kaeg Dum, (2) Co Coa Karn Dum and (3) Taiwan and Solo. She stated that Kaeg Dum cultivar has a close genetic relationship with Kaeg Nuan, Taiwan with Solo and Co Coa Karn Dum with Mexico-America. Therefore, there were only a few varieties of papaya cultivars have been used by growers within the past decades. Thus diversity in genetic traits among papaya cultivars was relatively small.
The results found with this study on the application of AFLP primers showed
that an increase in DNA bands was attained with 55 pairs of primers and 9 pairs
were not able to produce their DNA bands. However, out of 55 pairs of primers
only 12 pairs were randomly chosen for this investigation and the results revealed
that polymorphic markers of bands reached a value of 66.08% (485 bands). The
results indicated a close relationship on genetic traits among the thirty papaya
cultivars used where polymorphic information contents (PIC) were distributed
into three ranges, i.e., 0.003-0.05 (48.45%), 0.15-0.20 and 0.35-0.40 (9.70%)
and 0.15-0.20 (2.47%). When it comes to the effect due to genetic traits on
similarity coefficients with the use of its dendrogram, it was found that the
thirty papaya cultivars could be categorized into six groups but their ranges
on similarity coefficients ranged from 0.728 to 0.920 only so this range of
values indicates a close relationship among the thirty papaya cultivars used.
The obtained results were similar to the study of Kim et al. (2000)
where they collected 71 accessions of papaya cultivars of commercial, improved
and unimproved breeding lines and able to identify the cultivars with the use
of 9 pairs of ALFP primers. They attained 186 markers with a highest value of
0.880 from their cluster analysis. They stated that papaya accessions being
used did not provide any information on the diversity of genetic traits. Furthermore,
they stated that self-pollinated hermaphrodite cultivars gave some considerable
levels of variations similar to those of open-pollinated cultivars. However,
they were able to differentiate the 71 accessions from other 6 Carica species
where identity of breeds of the 6 species was not similar to their 71 accessions.
Similarly, Droogenbroeck et al. (2002) carried out papaya experiment
to identify papaya breeds in Ecuador with the use of AFLP markers reported that
from 95 papaya accessions, the papaya plants produced 491 markers where these
markers derived from 5 pairs of random primers and the results on cluster analysis
revealed that the papaya accessions used had its close relationship on genetic
traits and the results did not agree with the results derived from taxonomic
Although the results on cluster analysis attained with this work signified
a close relationship among the thirty papaya cultivars but still the papaya
cultivars could be categorized into six groups according to their similarity
coefficients of the dendrogram figure. The results evidently showed that the
thirty papaya cultivars could be differentiated into six different groups in
spite of their close relationship on genetic traits. This must be attributable
to perhaps (1) the limited varieties of papaya imported to Thailand in the past
decades or perhaps even now and then, (2) Seeds of productive cultivars must
have been always kept by growers for their own uses from generation to generation,
(3) natural selection and natural hybridization of papaya cultivars due to the
mixing up or a closed planting distance among papaya plants of different breeds
could have its effect on the mixing up of their genetic traits from generation
to generation and (4) another reason could have been due to a failure in breeding
programme and selection of the scientists within the country when some large
amounts of investment budget must be used in order to produce new cultivars
for use, (5) lack of well trained plant breeders of papaya crop or perhaps scientists
ignore this prospect due to plentiful amount of papaya fruits available in the
markets. Therefore, it is an urgent need for the Thai scientists both plant
breeders and plant physiologists to pay more attention to this particular cash
crop in order to provide both productive varieties and technologies of know
how for prospective growers in the country since high quality papaya fruits
could be exported annually and some considerable amounts both fresh and ripe
are needed for domestic consumption, particularly in all large cities and even
hotels and restaurants and etc.
The author wishes to thanks the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency of Thailand for financial assistance. Srisaket Horticultural Research Center, Loei Highland Horticultural Station, Phichit Horticultural Research Center, Department of Horticulture, Kaset Sart University, Khon Kaen Horticultural Research Center, Department of Horticulture, Ubon Ratchathani University for seeds of papaya cultivars used in the experiment, Staff members of the Department of Horticulture, Ubon Ratchathani University for their kind assistance during this research was carried out.
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