Indonesian Journal of Forestry Research Vol.
9 No.
April 2022, 63-72
ISSN: 2355-7079/E-ISSN: 2406-8195
GENETIC RELATIONSHIP OF SEVERAL MORPHOLOGICAL AND
MOLECULAR CHARACTERISTICS OF Phalaenopsis amabilis (L.
Blume ORCHIDS FROM THE MERATUS MOUNTAINS OF SOUTH KALIMANTAN.
INDONESIA
Dindin H.
Mursyidin*.
Rubiansyah, and Badruzsaufari Laboratory of Genetics and Molecular Biology.
Faculty of Mathematics and Natural Sciences.
Universitas Lambung Mangkurat.
South Kalimantan, 70714.
Indonesia Received: 16 January 2021.
Revised: 24 January 2022.
Accepted: 15 April 2022
GENETIC RELATIONSHIP OF SEVERAL MORPHOLOGICAL AND MOLECULAR
CHARACTERISTICS OF Phalaenopsis amabilis (L.
) Blume ORCHIDS FROM THE MERATUS MOUNTAINS OF SOUTH KALIMANTAN.
INDONESIA.
Phalaenopsis amabilis (L.
) Blume orchid is one of the most popular orchid species in the world.
However, this ornamental plant is threatened in its natural habitat, the Meratus Mountains of South Kalimantan.
Indonesia.
This study determines and analyzes the genetic relationship among several morphological characteristics of P.
amabilis from this region combined with molecular (RAPD) markers.
A total of ten orchid samples comprised of nine moth orchids (P.
and one species (P.
cornu-cerv.
as an outgroup, as well as ten RAPD primers were used in this Based on the morphological markers, the moth orchids have a moderate level of genetic diversity, indicated by Shannon's index value of 0.
In contrast to molecular markers, this germplasm shows high genetic polymorphism, shown by the polymorphism degree of 100% for all primers used.
The cluster analysis shows that this germplasm can be divided into two clusters for morphological and five for molecular Following these markers, the grouping of moth orchids was nearly corresponding to their origin.
Thus, this information could be useful as a reference for orchid conservation and breeding programs in the Keywords: Breeding and conservation, genetic polymorphism, orchid.
Phalaenopsis KEKERABATAN GENETIK ANTARA BEBERAPA KARAKTER MORFOLOGI DAN MOLEKULER
DARI ANGGREK
Phalaenopsis amabilis (L.
) Blume ASAL PEGUNUNGAN MERATUS.
KALIMANTAN SELATAN.
INDONESIA.
Phalaenopsis amabilis (L.
) Blume merupakan salah satu jenis anggrek terpopuler di dunia.
Namun tanaman hias ini telah terancam di salah satu habitat aslinya, yaitu Pegunungan Meratus.
Kalimantan Selatan.
Indonesia.
Tujuan penelitian ini adalah untuk menentukan dan menganalisis kekerabatan genetik dari beberapa karakter morfologi dari P.
amabilis dari wilayah tersebut dan menggabungkannya dengan penanda molekuler (RAPD).
Sebanyak sepuluh sampel anggrek, terdiri atas sembilan anggrek bulan (P.
dan satu spesies outgroup (P.
cornu-cerv.
, serta sepuluh primer RAPD telah digunakan dalam penelitian ini.
Berdasarkan penanda morfologi, anggrek ini memiliki tingkat keragaman genetik sedang, ditunjukkan dengan nilai indeks Shannon sebesar 0,5.
Berbeda dengan penanda molekuler, plasma nutfah ini menunjukkan variasi genetik yang tinggi, ditunjukkan dengan derajat polimorfisme sebesar 100% untuk semua primer yang digunakan.
Hasil analisis klaster menunjukkan bahwa plasma nutfah ini terbagi menjadi dua kelompok utama untuk penanda morfologi dan lima kelompok untuk penanda molekuler.
Berdasarkan kedua penanda, pengelompokan plasma nutfah ini relatif berkaitan dengan wilayah asalnya.
Dengan demikian, informasi ini diharapkan dapat digunakan sebagai acuan untuk program konservasi dan pemuliaan anggrek pada masa Kata kunci: Pemuliaan dan konservasi, variasi genetik, anggrek.
Phalaenopsis Corresponding author: dindinhm@gmail.
A2022 IJFR.
Open access under CC BY-NC-SA license.
doi:10.
20886/ijfr.
Indonesian Journal of Forestry Research Vol.
9 No.
April 2022, 63-72
INTRODUCTION
The Meratus Mountains of South Kalimantan Province.
Indonesia, is one of the diversity centers of native orchids in the In this region, hundreds of native orchids germplasms are currently threatened by natural and human impacts, making it difficult to find in their customary habitat (Muslimah.
Rachmawaty.
Hoesain.
Ninsyh, & Yulianto.
Even though some of them have been categorized as endangered species by CITES (Committee of the International Trade of Endangered Specie.
One orchid species that is very difficult to find in their customary habitat is the moth orchid (Phalaenopsis amabilis (L.
) Blum.
The local people of this region recognized three forms of the orchids, namely the AoPelaihariAo from the Birah Bajuin Mountain.
Tanah Laut Regency, the AoMeratusAo from the Hulu Sungai Selatan Regency, and the AoHalongAo from the Balangan Regency of South Kalimantan (Rusmayadi.
Sumardi.
Sudjatmiko, & Kuswidyosusanti, 2.
For some orchid collectors, these three forms are very difficult to distinguish morphologically.
Hence, further verification using molecular markers is very The moth orchid of AoPelaihariAo is one of the most famous orchids in the world (Muslimah et al.
, 2.
This moth orchid is generally recognized based on the flower characteristics, both shape, size, motif, and length of the flowering period (Tsai.
Chou.
Wang.
Ko.
Chiang, & Chiang, 2.
In general, the flower of the AoPelaihariAo orchid is white, decorated with a yellow-brown spot motif on the labellum, and has flowers with florets reaching more than 50 units on each stalk.
The orchid stalk could reach 80 cm, the length of the flowering period is up to 6 months (Muslimah et al.
, 2.
Consequently, it is not surprising that this orchid is the most searched and is very popular with collectors and breeders around the world.
In 2009, this moth orchid was even designated as one of IndonesianAos national flowers or AuPuspa PesonaAy .
n Indonesian term.
making it a national superior variety or the best cross-
ISSN: 2355-7079/E-ISSN: 2406-8195
parent material for the breeding program (Muslimah et al.
, 2.
In Indonesia, the moth orchids is spread across several large islands, such as Java.
Sumatra.
Sulawesi.
Kalimantan, and Papua (Fatimah & Sukma, 2.
However, the existence of the moth orchids in Meratus Mountain of the South Kalimantan Province is more worrying than the others, as mentioned Thus, the conservation and breeding programs of the orchid are very urgent to The Indonesian Orchid Association of South Kalimantan branch has carried out some activities to conserve and preserve this While these efforts have not been optimally carried out, some activities showed less satisfactory results (Muslimah et al.
, 2.
The limited information about orchid genetics is one of the obstacles to these two activities.
In general, the conservation and breeding tasks involve several principal activities, one of which is identifying and characterizing germplasm with a comprehensive study .
an de Wouw, van Hintum.
Kik, van Treuren, & Visser, 2.
According to Pellens and Grandcolas .
, critical and accurate information about germplasm sources is urgently needed to support these programs.
Hence, the objective of our study was to determine and analyze the genetic relationship among several morphological and molecular characteristics of this orchid, particularly by RAPD/Random Amplified Polymorphic DNA While these markers have certain limitations, a combination of these two markers may represent a comprehensive feature of the genetic diversity of this germplasm (Rocha et al.
Until now, many researchers have been using these markers for asAsessing the genetic diversity of various germplasms, like orchids (Khoddamzadeh.
Sinniah.
Kadir, & Kadzimin.
In Indonesia.
RAPD has been used for analyzing the genetic diversity of orchids but is still very limited.
Moreover, the orchid samples are the hybrids, not from the natural areas, as employed by Sulistianingsih and Purwantoro .
Thus, the results of our study are Genetic Relationship of Several Morphological and Molecular Characteristics of Phalaenopsis .
(Dindin H.
Mursyidin et al.
valuable in supporting orchid conservation and breeding programs in the future.
(Figure .
Specifically, for the hybrid orchid outgroup samples were obtained from the Food.
Agriculture, and Fisheries Service Agency of Banjarmasin City and an orchid collector in Banjarbaru regency.
South Kalimantan.
For morphological analysis, samples were directly observed at the sampling locations.
For molecular analysis, orchid leaves were sampled and put into a plastic bag containing silica gel.
II.
MATERIAL AND METHOD
Plant Materials A total of ten orchids samples with several morphological characteristics (Table .
, comprising nine moth orchids (P.
species and one of the deer-antlered orchid cornu-cervi .
s an outgrou.
were collected randomly from three locations of the Meratus Mountains, including three regencies of South Kalimantan.
Indonesia, namely Tanah Laut (Tal.
Balangan and Hulu Sungai Selatan (HSS) Table 1.
List of orchid samples employed in the study Local Name
Anggrek bulan AoPelaihariAo
Anggrek bulan AoHalongAo
Anggrek bulan AoHalongAo
Anggrek bulan AoHalongAo
Anggrek bulan AoHalongAo
Anggrek bulan AoMeratusAo
Anggrek bulan AoHalongAo
Anggrek bulana
Anggrek bulana
Anggrek bulan gergajib
Code
PA-01
PA-02
PA-03
PA-04
PA-05
PA-06
PA-07
PA-08
PA-09
Morphological Observation The morphological observations were carried out directly at the sampling locations on six morphological characters, those were:
resilience of flowers, thickness of the petals, length of the flower stalk, number of blossoms Scientific Name cornu-cervi Origin (Regenc.
Tanah Laut Balangan Balangan Balangan Balangan Hulu Sungai Selatan Balangan Tanah Laut Tanah Laut Tanah Laut Remarks: ahybrid, as comparison ban out group Figure 1.
A map of South Kalimantan showing the three sampling locations .
long the Meratus Mountain.
, where the moth orchids were collected: The regency of Tanah Laut (A).
Hulu Sungai Selatan (B), and Balangan (C) Indonesian Journal of Forestry Research Vol.
9 No.
April 2022, 63-72 per stalk, and visibility of veins on leaves, as well as leaf shapes.
Molecular Analysis Molecular analysis of the orchid was conducted in the Laboratory of Genetics and Molecular Biology.
Faculty of Mathematics and Natural Sciences.
Universitas Lambung Mangkurat.
Indonesia.
The activities started with the extraction of DNA by the DNAzol manufacturerAos The DNA was then quantified and measured by UV-Vis spectrophotometer at 260 nm wavelengths.
Amplification of this genetic material was conducted using 10 of the 22 selected RAPD primers (Table .
(Sulistianingsih & Purwantoro, 2.
with a total volume of 20 AAl, consisting of 17 AAl master mix PCR .
ontaining deionized water.
PCR buffer.
dNTPs.
MgCI2.
Taq DNA polymeras.
, 1.
5 ng/ AAL of each primer .
5 ng/ AAL of genomic/template DNA.
This reaction was performed by using Thermal Cycler PCR (Techne.
TC3000G.
USA) with a cycling condition (Mursyidin & Daryono, 2.
: initial denaturation at 94AC for 5 min.
denaturation at 94AC for 30 secs, annealing at 37AC for 30 secs, and extension to 72AC for 5 min .
hese stages were repeated for 45 and a final extension at 72AC for 7 min.
The amplified DNA was separated by 1.
of agarose gel electrophoresis and 1X TBE
ISSN: 2355-7079/E-ISSN: 2406-8195
H 8.
as a supporting medium.
These samples were stained by nucleic acid gel stains (GelRed.
Biotium.
USA) and observed with the DNA ladder .
Vivanti.
Observation of DNA fragment of each primer which was generated conducted by UV transilluminator and digital camera (Nikon Coolpix L.
Data Analysis Data were analyzed both for morphological and molecular markers.
Data analysis was started with scoring and standardizing the obtained Shannon-Weaver diversity index (HA.
was used to determine the phenotypic diversity of this germplasm.
Diversity indices were calculated based on phenotypic frequency using the standardized Shannon-Weaver Diversity index formula (Rabara.
Ferrer.
Diaz.
Newingham.
Ma, & Romero, 2.
Multivariate statistical analyses of characterization data were conducted using cluster analysis.
This analysis was done using the MVSP ver.
1 software (Kovach, 2.
The distance matrix was generated using the Euclidean Distance Coefficients and used as input for clustering using the unweighted pair group of arithmetic means (UPGMA) method.
For molecular data, each DNA fragment that develops at a particular rate of electrophoresis gel was measured by using a linear regression equation and considered as a single locus.
Hence, the same DNA fragments of some individual plants were interpreted as one homologous Table 2.
Selected RAPD primers employed in the locus.
The locus was then converted into binary matrix data by scoring the value of one .
if there is a DNA fragment and zero .
if there is Sequences GC content Primers no DNA fragment.
The binary matrix data was .
'-3') (%) then derived into a genetic distance matrix.
OPA-02
TGCCGAGCTG
OPA-04
AATCgCTG
calculate the genetic distance of the genotype OPB-01
GtCGCTCC
pairs found in different individuals, the Dice
OPB-06
TGCTCTGc coefficient was applied.
Based on the value of OPB-07
GGTGACGCAG
genetic similarity then the clustering analysis OPS-12 CTgTGAGT was conducted.
The clustering analysis and OPA-09
gTAACGCC
reconstruction of a phylogenetic relationship
OPA-10
GTGATCGCAG
of this germplasm were performed using the OPB-05
GATGACCGCC
UPGMA with the assistance of the NTSys ver.
OPB-10
CTGCTgAC 2 (Rohlf, 2.
A bootstrap analysis was also Source: Sulistianingsih and Purwantoro .
Genetic Relationship of Several Morphological and Molecular Characteristics of Phalaenopsis .
(Dindin H.
Mursyidin et al.
performed to evaluate the internal nodes on the i.
RESULTS AND DISCUSSION
Morphological Characteristics Although the three forms of orchids from the Meratus Mountains appear to be the same .
elatively difficult to distinguis.
morphologically (Figure .
, the results of further observations of some of these characters show quite significant differences (Table .
For example, based on leaf shape, the three forms of orchids could be distinguished because the leaf shapes are different, namely ovate for AoPelaihariAo, elliptic for AoMeratusAo, and lanceolate for AoHalongAo (Table .
Other differences appear in the leaf veins and thickness of petals, whereas the AoPelaihariAo orchids has visible leaf veins and thin petals (Table .
Based on Table 3, it is also seen that AoPelaihariAo orchids have different characteristics compared to the other two orchids found in the Meratus Mountains, namely a long stalk of the flower with 9-17 blooms, a long resilience of the flower blooms .
-6 month.
, and a yellow-brown spot at the labellum, as well as a V-shape at the end of the The complete information on the differences in morphological characters of the orchids was presented in Table 3.
In brief, morphological observations reveal the unique feature of the three forms of moth orchids found in the Meratus Mountains.
The unique feature is characterized by the resilience of flowers bloom and the number of blossoms per stalk, as well as the shape of the observed leaves (Table .
According to van Figure 2.
The morphological features of the moth orchid flowers (A-D) and its plant habitus (E-H).
Remarks: A = P.
amabilis AoHalongAo.
B = P.
amabilis AoMeratusAo.
C = P.
amabilis AoPelaihariAo.
D = P.
amabilis hybrid, as a Table 3.
The morphological differentiation of the moth orchids endemically from the Meratus Mountains.
South Kalimantan, including the hybrid .
Traits observed The resilience of flowers bloom The thickness of the petals Length of the flower stalk Number of blossoms per stalk Visibility of veins on leaves Leaf shape AoHalongAo A 4-5 months Thick A 1.
5-15 unit Invisible Lanceolate AoMeratusAo A2 months Thick A 1.
5-10 unit Invisible Elliptic AoPelaihariAo A 4-6 months Thin A 1.
9-17 unit Visible Ovate AoHybridAo < 2 months Thick A 1.
5-10 unit Invisible Lanceolate Indonesian Journal of Forestry Research Vol.
9 No.
April 2022, 63-72 Huylenbroeck .
, the uniqueness of this germplasm is also based on the triangular shape of the labellum and the color sharpness of the Interestingly, based on this study, the AoPelaihariAo shows distinct flower formation.
number of florets in one stalk, and the length of the flowering period is more striking than the other two orchids found in the region.
So, it is not surprising that in 2009 this orchid had obtained formal legality from the Indonesian minister of agriculture as a superior national cultivar (Muslimah et al.
, 2.
According to Anumalla.
Roychowdhury.
Geda.
Mazid, and Rathoure .
, although morphological marker has a weakness because it is strongly influenced by environmental factors, breeders are using this marker to evaluate germplasm in the early stages of plant growth and development.
In other words, morphological analysis is still used in determining the genetic diversity of germplasm.
In genetics, the morphological marker or phenotype is the results of the expression of genotype which is influenced by environmental Thus, further verification of the germplasm studied using more stable molecular markers on environmental factors is very Furthermore, a combination of the ISSN: 2355-7079/E-ISSN: 2406-8195 two markers .
orphological and molecula.
is expected to produce a more comprehensive feature of the genetic diversity of germplasm (Rocha et al.
, 2.
Genetic Diversity Based on the morphological markers, the moth orchids of the Meratus Mountains have a moderate level of genetic diversity, indicated by Shannon's index value of 0.
5 (Table .
However, two of the morphological traits observed in this study were shown to have a high level of diversity, i.
, the resilience of flowers bloom and the number of blossoms per stalk, with an index of 0.
92 each.
Table 4.
Genetic diversity (HAo inde.
of the moth orchid based on morphological traits Morphological traits The resilience of flowers bloom The thickness of the petals Length of the flower stalk Number of blossoms per stalk Visibility of veins on leaves Leaf shape Average H' index Remarks: alow, bmoderate, chigh Table 5.
Polymorphism degree of the moth orchids from the Meratus Mountains of South Kalimantan, including their number and size of amplified DNA fragments generated by RAPD markers Primer
OPA-02
OPA-04
OPB-01
OPB-06
OPB-07
OPS-12
OPA-09
OPA-10
OPB-05
OPB-10
Total/Average The size range of amplified DNA fragment .
Total of DNA fragment .
Number of polymorphic DNA Polymorphism (%) Genetic Relationship of Several Morphological and Molecular Characteristics of Phalaenopsis .
(Dindin H.
Mursyidin et al.
Figure 3.
Visualization of amplified DNA fragments generated by four representative RAPD markers used in this study Remark: A = OPA-02.
B = OPB-06.
C = OPB-01.
D = OPB-07.
M = 100 bp DNA ladder (Vivanti.
Lane 1 = P.
amabilis AoPelaihariAo.
Lane 2-5, 7 = P.
amabilis AoHalongAo.
6 = P.
amabilis AoMeratusAo.
8-9 = P.
amabilis hybrid.
10 = P.
cornucervi, an outgroup In contrast to molecular markers, this germplasm shows high genetic diversity, shown by the polymorphism degree of 100% for all primers used (Table .
According to Mursyidin and Daryono .
, genetic diversity can be described using polymorphism, average heterozygosity, and allelic diversity.
In RAPD, this genetic diversity or polymorphisms are analyzed by measuring the presence vs.
absence of random amplified DNA fragments (Mursyidin and Daryono, 2.
In this case, the moth orchids of the Meratus Mountains of South Kalimantan, showed a unique profile of DNA fragments generated by RAPD markers (Figure .
In general, a total of 192 DNA fragments .
have been generated by these primers, where each primer resulted in a different number and size of DNA fragments (Table .
Furthermore.
OPA-10 and OPB-05 produced the lowest number of DNA fragments .
, whereas OPB-06 was the highest .
The differences in the number and size of DNA fragments in PCR may depend on the attachment site of a DNA primer to the genome of the sample (Clark & Pazdernik.
It means, that each primer has a specific sequence, and the attachment site must be different (Clark & Pazdernik, 2.
These differences are influenced by other factors as well, such as the concentration of MgCl2.
DNA template, and DNA polymerase, as well as PCR annealing temperature (Dominigues, 2017.
Maddocks & Jenkins, 2.
Siddiqi and Nollet .
stated that the presence of polymorphic DNA fragments in a genome reveals the genetic diversity of germplasm.
In other words, polymorphic DNA fragments explained the genetic status of germplasm in the population (Chenu, 2.
Genetic Relationships The cluster analysis shows that this orchid germplasm is divided into different groups, two for morphological (Figure .
and six for molecular markers (Figure .
According to Ewens .
, these differences may be caused by several factors, such as the evolution and adaptation to the local conditions, crossbreeding, population history, speciation, population distribution, and gene flow (Ewens.
Following the morphological markers, the moth orchid of AoMeratusAo was joined by the hybrid in the first cluster, whereas AoHalongAo and AoPelaihariAo were in the second.
For the molecular Indonesian Journal of Forestry Research Vol.
9 No.
April 2022, 63-72
ISSN: 2355-7079/E-ISSN: 2406-8195
Figure 4.
Relationship of the moth orchids from the Meratus Mountains of South Kalimantan, based on morphological characters Figure 5.
Relationship of the moth orchids from the Meratus Mountains of South Kalimantan, based on RAPD analysis.
Note.
percentage on nodes were generated by bootstrap 1,000 replicates marker, the moth orchid of AoPelaihariAo was separated from other samples and formed a single group (Cluster I).
The AoHalongAo moth orchids were grouped into Cluster II, except for one sample which was grouped to Cluster i, and joined with the AoMeratusAo.
The hybrids (Clusters IV & V) formed a closely related group to an outgroup.
Following the dendrograms (Figures 4 & .
, the clustering of moth orchid germplasm was near corresponding to their geographic origins.
According to Cotrim.
Monteiro.
Sousa.
Pinto, & Fay .
, the pattern of this relationship may be caused by hybridization and genetic introgression .
epeated back-crossing ).
In this context, hybridization may accelerate speciation via adaptive introgression, or cause nearinstantaneous speciation by allopolyploidization (Cotrim et al.
, 2.
In brief, these two relationships .
also represented a linearization or congruency between morphological and molecular markers.
While this congruence needs further verification, some researchers have shown this matter.
For example, for forage palm genotype (Rocha et al.
, 2.
, neotropical passerine (Garcia.
Barreira.
Lavinia, & Tubaro, 2.
, and Calochromini (Motyka.
Masek, and Bocak, 2.
Further, a comprehensive analysis of this orchid relationship has been done by several researchers, for example, by Tsai.
Chiang.
Huang.
Chen, & Chou .
using the internal transcribed spacer (ITS) sequences.
Based on their study.
amabilis is incorporated in the same cluster as P.
sanderiana, whereas P.
cornucervi is closely related to P.
Based on microsatellite markers.
amabilis is closely Genetic Relationship of Several Morphological and Molecular Characteristics of Phalaenopsis .
(Dindin H.
Mursyidin et al.
related to P.
fuscata (Fatimah & Sukma, 2.
Using the RAPD marker.
Niknejad.
Kadir.
Kadzimin.
Abdullah, and Sorkheh .
reported that P.
amabilis has a close relationship with P.
hieroglyphica, whereas P.
cornu-cervi with P.
mannii and P.
Based on the results of our study.
amabilis has a distant relationship with P.
cornu-cervi.
Finally, this information may be valuable for improving the efficiency and effectiveness plant conservation and breeding programs in the future (Flint-Garcia, 2013.
Pellens & Grandcolas, 2016.
Singh, 2.
For conservation, information on genetic relationships can apply in inferring species and their evolutionary history, including helping analyze species delimitation, gene flow, and genetic differentiation.
In other words, the use of this relationship is of current interest given its objective metrics for conservation in the past evolution history, the present genetic status of species, and management for future ones (Fernyndez-Garcya, 2.
For plant breeding programs, similar information can be used in predicting the genetic diversity of the offspring when individuals cross (Acquaah, 2012.
TurnerHissong.
Mabry.
Beissinger.
Ross-Ibarra, & Pires, 2.
IV.
CONCLUSION
In this study, morphological and molecular markers provided a unique feature of the genetic relationship of the moth orchids of the Meratus Mountains of South Kalimantan.
Indonesia.
Based on the cluster (UPGMA) analysis, this germplasm is divided into two .
or morphological marker.
and five groups .
or molecular one.
The results indicate that the grouping of orchid germplasm has quite corresponded to geographic location.
While the results provide valuable information to support orchid conservation and breeding in the future, it requires further verification, especially using more accurate molecular markers, such as SNP.
ACKNOWLEDGEMENT
The authors wish to thank the Head of the Food.
Agriculture, and Fisheries Service Agency.
Banjarmasin City.
Indonesia for providing the P.
amabilis hybrid and P.
cornu-cervi as a comparison and outgroup, respectively.
The author also wishes to thank RifaAoi and Zia for helping with samples collection and molecular analysis in the laboratory.
REFERENCES