MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESEVALUATION OF GENETIC DIVERSITY OF TEA TREE Melaleuca alternifolia COLLECTED FROM DONG
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Figure 2.1 The tea tree (Ti he picture was taken at the Dong Ti hap Muoi conservation.)
Melaleuca alternifolia, commonly known as tea tree, is an Australian native plant belonging to the Myrtaceae family It flourishes in various environments, including streams, wetlands, clay soils, saline coastal areas, marshes, and highland soils, particularly in southeast Queensland and New South Wales This versatile plant is the primary source for the Australian Melaleuca essential oil industry.
The tea tree is a versatile shrub that can grow up to 7 meters tall and features distinctive white, paper-like bark Notably, Melaleuca can be harvested for the first time just 1 to 2 years after planting, allowing for annual bud harvests for over 20 years This remarkable characteristic makes it an economically efficient and stable crop choice.
Based on morphological variety, the morphological features of tea trees are defined and because of challenging environmental circumstances and _ other considerations specific descriptions of certain characteristics are provided below.
The leaves of the tree are alternately arranged, measuring 10-35 mm in length and 1 mm in width, characterized by their soft, smooth, and pointed appearance, with visible oil glands rich in essential oils Typically, trees older than three years bloom in October and November, producing white to cream flowers that cluster in the leaf axils or at branch ends, creating a soft aesthetic Each leaf axil features a single flower with a tubular sepal up to 3 mm long, a 2-3 mm long white corolla, and a pistil measuring 3-4 mm The inflorescences consist of dense clusters of flowers, while the tree's fruit is a woody capsule resembling a mangosteen, containing several tiny seeds that disperse among the branches.
2.1.3 Introduction to tea tree essential oil
Tea tree oil is classified into three groups based on its chemical composition: Group 1 contains low 1.8-cineole (3%) and high terpinen-4-ol (45.4%), Group 2 has medium 1.8-cineole (30.3%) and low terpinen-4-ol (1.7%), while Group 3 features high 1.8-cineole (64.1%) (Williams et al., 1989) Australia is the leading producer of essential oils rich in terpinen-4-ol, making it the most valuable category (Kha et al., 2018) The composition of tea tree essential oil is influenced by various geographical, climatic, and biological factors along the coast.
Tea tree oil, abundant in terpinen-4-ol, is a versatile ingredient that is safe for the skin and widely used in various products such as mouthwash, cosmetics, perfumes, shampoos, scented soaps, and whipped creams Additionally, it is effective in treating common skin conditions, including acne, warts, boils, herpes, burns, insect bites, nail fungus, athlete's foot, and sweaty feet.
Melaleuca plants have been sent to Vietnam for experimentation since 1993 (A.S.
Melaleuca trees in Vietnam and Australia exhibit similar physical characteristics, with tea tree pilot models in Northern Vietnam producing essential oils containing terpinen-4-ol levels between 33% and 43% The production and application of these oils are expanding, but caution is advised with cajuput essential oil due to its toxicity, despite no recorded fatalities (Nguyen et al., 2019) According to the Australian Standard (AS 2782-1985), cajuput essential oil must contain at least 30% terpinen-4-ol, a recognized antibacterial agent, while the skin irritant 1,8-cineole should not exceed 15% in tea tree products (Carson et al., 2006).
Tea Tree is grown in the Moc Hoa and Thanh Hoa districts of Long An Province, thriving on alum soil When proper cultivation techniques are employed, these trees can withstand high levels of alum and continue to flourish even in dry conditions.
In the Thanh Hoa district, there are 10 hectares dedicated to tea plants, while approximately 15 hectares of tea trees have been cultivated in the Dong Thap Muoi conservation area of Moc Hoa district to ensure pharmaceutical supplies The Moc Hoa Tram Factory produces high-quality tea tree essential oil that meets Good Manufacturing Practices (GMP) standards One of the two advanced scientific initiatives being implemented in the Conservation Area focuses on the Melaleuca tea tree.
2.2 Analysis of genetic diversity based on molecular markers
2.2.1 The concept of genetic diversity
Genetic diversity is all the different genetic makeup of individual plants, animals,fungi and microorganisms There is genetic variety both within and between species.
Genetic diversity refers to the range of genetic variations that can be inherited within a population, among different populations, and between individuals of the same species as well as across various species (L.A et al., 2012).
Genetic diversity refers to the variety of heritable variants found within a species, among communities, or between different species This diversity arises from variations in the sequence of the four essential nucleotide bases and the composition of the nucleic acids that constitute the genetic code.
Genetic variations among individuals within the same breed or across different breeds can influence external features To uncover these genetic differences, researchers often seek specific markers that indicate these variations (Bui Chi Buu et al., 1999).
2.2.2 A few techniques for researching genetic diversity
Morphological features serve as key indicators for identifying genetic variation, as specific genes produce proteins that manifest in observable traits These morphological indicators can effectively differentiate individuals exhibiting the same characteristic (Carreira et al., 2016).
While morphological markers are commonly used to assess genetic diversity, they have significant limitations, as an individual's hereditary genes may not always be reflected in their phenotypes Additionally, these markers are highly influenced by environmental factors, making them less reliable than DNA analysis The complexity of certain species, such as those within genera, is further complicated by frequent occurrences of crossings, apomixes, polyploids, and mutant buds, leading to ambiguous taxonomic classifications Consequently, morphological indicators are often employed alongside more precise genetic measures.
Combining molecular markers with biochemical traits enhances the accuracy of genetic diversity assessments compared to relying solely on morphological indicators, which can be influenced by environmental factors Complex species, especially within certain genera, often experience frequent crossings, apomixes, polyploidy, and mutant buds, leading to taxa in amorphous stages (Kumar et al., 2010) Therefore, integrating morphological traits with molecular markers is essential for improving the precision of genetic diversity evaluations in these species.
Modern techniques have significantly improved the assessment of plant genetic diversity, making it easier, more cost-effective, reliable, and quicker than traditional methods Utilizing molecular biology, researchers construct DNA molecular markers, which are essential for studying genetic diversity These techniques can be categorized into two major groups, incorporating a variety of trustworthy and widely-used molecular markers.
PCR-based techniques: AFLP, RAPD, SSR, ISSR.
Technique based on DNA - DNA hybridization technique: typically RELP
2.2.3.1 AFLP technique (Amplified Fragment Length Polymorphism)
Some domestic and international research on Melaleuca alternifol1a
In Vietnam, there is a lack of research on the genetic diversity of tea trees utilizing ISSR markers A study conducted by Tran Thi Ngoc Tram and colleagues evaluated the genetic diversity of 49 Melaleuca samples collected from various locations in Thua Thien Hue.
(2021) utilized the RAPD approach demonstrating that the genetic similarity across groups which ranges from 0.891 to 0.963 is fairly high.
The ISSR marker has not been utilized in international studies to assess the genetic diversity of tea trees Research indicates that while there is limited variation among Melaleuca populations in Australia, a significant portion (42%) of genetic diversity can be linked to regional differences, particularly in Queensland and New South Wales The genetic differences observed between these groups correlate with their geographic isolation In a study involving 500 Melaleuca alternifolia individuals, five SSR primers were employed, resulting in 98 bands and a 90% polymorphism level This research highlights both widely dispersed planting patterns and populations exhibiting low levels of inbreeding.
2003) Only 2 populations, Queensland and New South Wales, constitute different genetic origins due to geographic isolation.
MATERIAL AND METHOID 5-5 + 2 S2 S221 291gr re 13 3.1 Research time o0 on
This graduate thesis had been done on 7/2023 - 10/2023 at A204 Molecular Biology, Research Institute for Biotechnology and Environment (RIBE), Nong Lam University Ho Chi Minh City.
50 tea tree leaf samples were collected at the Dong Thap Muoi Center for Research, Conservation and Development of Medicinal Materials, Long An province as research materials.
Figure 3.1 Representative tea tree leaf sample (A) was sample TT02, (B) was detailed photo of the sample TT40.
Table 3.1 List of 50 tea tree samples collected at Dong Thap Muoi Medicinal Materials Conservation Center, Long An province used for research
Table 3.1.(Continue) List of 50 tea tree samples collected at Dong Thap Muoi
Medicinal Materials Conservation Center, Long An province used for research
3.2.2.1 Evaluation of leaf morphological marker
To collect samples, select several numbered trees from the garden and gather 2 to 3 branches from each Place each sample in a separate zip bag, clearly labeling it with the sample name and collection time After transporting the samples, store them in the refrigerator for extraction and evaluation of leaf morphological characteristics.
To analyze leaf morphology, focus on essential parameters such as leaf length, shape, and the color variations between young and old leaves Utilize a ruler for accurate measurements and observations to evaluate leaf color and structure Record the measurements of key variables, including leaf length and breadth, and document the findings systematically.
3.2.2.2 Sample extraction and DNA testing of Melaleuca alternifolia leaf
Young leaf samples were subjected to a DNA extraction process developed at RIBE, Nong Lam University, HCM City
For optimal sampling, utilize young leaf samples, which should be collected and stored in zip bags in a refrigerator Following extraction, these samples must be preserved in a freezer at -20°C to maintain their integrity.
To begin the extraction process, place a young leaf into a 1.5 mL Eppendorf tube and add 300 µL of extraction buffer, which includes 200 mM Tris (pH 8), 25 mM EDTA, 250 mM NaCl, and 0.5% SDS Homogenize the mixture thoroughly with a plastic pestle until a smooth consistency is achieved, then proceed with pipetting using a Nichiryo pipette from Japan.
100 uL — 1000 kh tip (Germany) to add an additional 300 uL of extraction buffer and mix thoroughly.
Step 2: Incubate the mixture in a heat block at 65°C for 40 minutes.
Step 3: After incubation, centrifuge the mixture using a centrifuge machine (Hettich — Germany) at 10.000 rpm for 5 minutes.
To transfer 400 pL of supernatant, utilize a pipette with a 100 pL - 1000 pL tip, and place it into a new Eppendorf tube Next, add an equal volume of PCI solution, which consists of Phenol, Chloroform, and Isoamyl alcohol in a 25:24:1 ratio, and ensure thorough mixing.
Step 5: Centrifuge at 10.000 rpm for 5 minutes Use a pipette to transfer 300 pL of the supernatant to a new Eppendorf tube.
Step 6: Add an equal volume of CI solution (Chloroform/Isoamyl alcohol in a ratio of 24:1) and mix well Then, centrifuge at 10.000 rpm for 5 minutes.
Step 7: Use a pipette to transfer 200 nL of the supernatant to a new Eppendorf tube Add 0.6V of Isopropanol solution and gently shake.
Step 8: Place the mixture in a -20°C freezer (Sanyo - Japan) for 30 minutes.
Step 9: After the freezing period, centrifuge the mixture at 12.000 rpm for 10 minutes and discard the supernatant.
Step 10: Rinse the DNA by adding 500 pL of 70% Ethanol, then centrifuge at 12.000 rpm for 3 minutes and discard the supernatant.
Step 11: Repeat step 10 once more.
Step 12: Air-dry the Eppendorf tube until no liquid is visible Add 50 uL of 1X
TE solution to the Eppendorf tube containing the DNA precipitate and store at - 20°C.
After the extraction process, DNA qualitative analysis will be performed.
3.2.2.3 Product qualification by electrophoresis technique
To prepare a 1% agarose gel, weigh 0.5 g of agarose and mix it with 50 mL of 0.5X Tris-Acetate-EDTA (TAE) solution Heat the mixture in an Electrolux microwave oven at 500W for one minute to fully dissolve the agarose, then allow it to cool for approximately fifteen minutes before use.
Pour the gel into the pre-installed 13-well comb tray Wait about 30 minutes for the gel to completely solidify Check the gel is hard enough, then remove the comb
Carefully extract the gel and position it in the electrophoresis tank (Biorad electrophoresis machine) with the well end facing the negative pole of the power source Then, add 0.5X TAE buffer to the gel pad.
Samples were prepared by mixing 10 µL of 5X loading dye with 5 µL of sample DNA before being injected into the wells The electrophoresis tank was sealed and connected to a power source, allowing the total DNA to be separated at a voltage of 100 V for 15 minutes Finally, the results were examined under UV light.
Electrophoresis results indicate that the extracted sample contains DNA, which is visualized as a distinct band on the gel pad after exposure to UV light Samples exhibiting clear ice will advance to the next stage of analysis.
In this study, we will utilize 20 ISSR primers listed in Table 3.2 to randomly select a DNA sample for PCR analysis The aim is to assess polymorphism and determine the optimal annealing temperature (Ta) by testing six different temperatures ranging from 50°C to 55°C The temperature yielding the clearest product will be selected for further analysis Primers that produce bright and clear bands with high polymorphism rates will be retained for subsequent ISSR-PCR reactions across all samples.
After being extracted and stored at -20°C, the DNA sample will be diluted 10 times to continue the PCR reaction.
A PCR reaction using ISSR primer was conducted in a total volume of 12.5 µL, comprising 6.25 µL of 2X Master mix from Meridian Bioscience, 0.5 µL of Sigma primer, a specified volume of sample DNA, and 4.75 µL of deionized water The mixing of DNA samples was carried out in a level 2 biological safety cabinet The PCR amplification was executed on an Applied Biosystems 2720 machine, following the thermal cycling protocol outlined in Table 3.3.
Table 3.2 List of 20 primers for PCR reaction - ISSR
No Primer name Sequence primer (5’-3’)
10 UBCS45 (CT)sRG lội UBC848 (CA)sAGG
R = (A, G); Y= (C, 1) (Behera et al, 2008; Anil et al, 2015)
Table 3.3 Thermal cycling for PCR reaction
The temperature (T°C) for PCR-ISSR reactions varies depending on the specific primer used The resulting products were analyzed through electrophoresis on a 1.5% agarose gel in TAE 1X, applying a voltage of 100V for 45 minutes A 1 kb DNA standard was utilized to determine the size of the amplified DNA.
18 fragments Then, read the results using a UV light cabinet and take pictures, then encode the data and use it for data analysis.
The results of the PCR reaction using the ISSR indicator were analyzed on a gel, and the data were subsequently transformed into a binary matrix in Excel In this matrix, the presence of a DNA band is denoted by "1," while the absence is indicated by "0." The file name is placed in the top row, and the first column of the second row contains the number "1" to represent generic data Column 2 records the total number of data rows, Column 3 counts the data columns followed by the letter "L," and Column 4 tallies the number of non-empty cells containing data.
The analysis utilized the Dice correlation coefficient for data grouping, while the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) facilitated the classification process Additionally, the Sequential Agglomerative Hierarchical Non-Overlapping (SAHN) method was employed to construct a genetic classification tree using NTSYSpc 2.1 software.
4.1.1 Results of analyzing the leaf morphology tea trees
In a study of 50 Melaleuca samples, key leaf morphological parameters were assessed, including tree height, triradius, leaf shape, length, breadth, and color in both young and old leaves The findings reveal that tea tree leaves are small, lanceolate, and arranged alternately along the branch Additionally, older leaves exhibit a tough, dark green appearance, while young leaves are characterized by a lighter green hue, often described as banana green.
Table 4.1 Records the leaf dimensions of 50 Melaleuca samples. leet von Bene
No (enn) Guinn’ radius Tree Feedback
(cm) (cm) TT01 0.8-1.6 0.5-1 80 200 Small leaves,narrow
TT03 0.6-2.2 0.6-1.2 70 180 Long leaves, little slight
TT05 1-18 06-11 77 (jj - ̣fitonlnweragz length, oval TT06 09-17 0.7-0.9 60 140 Leaves are of average length, narrow
TT07 1-18 05-12 62 fa ô6 nee alam length, little slight TT08 12-25 0.9-1.9 63 155 Long leaves, oval
Leaves are of average TT09 11-24 0.9-1.9 69 165 lgrgôx, fictte alight
TTI0 11-21 07-1 54 160 nơi, ate Ob SEER
TT11 1-1.7 0.5-1 60 185 Small leaves, narrow TT12 1-2 0.5-0.9 71 210 Long leaves, narrow
Table 4.1.(countine) Records the leaf dimensions of 50 Melaleuca samples.
No a tau radius Tree Feedback
TT14 1-2.5 0.8-1.3 52 175 Long leaves, little slight TT15 1.3-2.1 0.6-1 67 150 Long leaves, little slight TT16 1.5-2.2 0.5-1 89 200 Long leaves, little slight TT17 1.6-2.5 0.9-1.5 87 220 Long leaves, oval
TT19 1.4-1.8 0.6-1 90 195 Medium leaves, narrow TT20 1-2 06-09 67 155 Leaves are of average length, narrow
Leaves are of average ial LS T8 LỔU tangs, Hele slight
TT22 L282 LL 65 —_ ee and the widest TT23 1.3-2.8 1.2-2 101 290 Long leaves, oval
TT26 L7 0451 42 ng | IS length, narrow
Leaves have the smallest TT28 0.5-1 0.5-0.8 30 100 aad tho sinh
TTS1 lig4 6712 67 [nu P6 8356101H60BQ8 length, narrow TT32 1-19 05-1 65 190 Leaves are of average length, narrow TT33 1.3-2.6 0.8-1.5 80 210 Long leaves,oval
TT34 12-2 05-] 63 185 Leaves are of average length, oval TT35 1.4-2.4 0.7-1.4 81 195 Long leaves, narrow
Table 4.1(countine) Records the leaf dimensions of 50 Melaleuca samples.
No a tau eatits ee Feedback
(cm) (cm) TT36 1-1.7 0.5-1 71 175 Small leaves, narrow T137 1-2 0.4-0.9 54 150 Small leaves, little slight TT38 1.3-2.3 1-2 72 195 Long leaves, little slight TT39 1.5-2.5 0.8-1.3 68 165 Long leaves, little slight TT40 13-22 0.6-1.1 64 165 Long leaves, little slight TT41 15-22 0.7-1.5 92 220 Long leaves,oval
TT44 14-18 © 05-1 73 145 ma TT45 2 GL 78 155 nth, rea
T148 1.2-2.4 0.7-1 75 185 Small leaves, narrow TT49 1.1-2.1 0.7-1.1 Hợi 195 Small leaves, narrow TT50 0.9-1.8 0.6-1.1 40 130 Small leaves, narrow