MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESEVALUATION OF GENETIC DIVERSITY OF Melaleuca cajuputi POPULATIONS AT DONG THAP MUOI CE
Conservation and, Development of Melaleuca cajuputi at Dong Thap Muoi Center
Melaleuca species demonstrate remarkable resilience to various environmental conditions, thriving in acidic, saline, and waterlogged soils, as well as arid lands They particularly flourish in peatlands and inundated areas like swamps, although they can also survive in brackish waters While drier conditions may stunt their growth and lead to curved structures, these plants continue to adapt and persist in less favorable environments.
In Vietnam, Melaleuca is scattered throughout three regions of the country, concentrated in the mountainous areas of Binh Tri Thien and the wetlands of the South.
M cajuputi thrives in degraded peat swamps, showcasing rapid growth due to its tolerance for waterlogged and acidic environments This species often forms pure forest communities, which can restrict the growth of other plants However, in drier fringe areas with less permanent inundation, M cajuputi coexists with diverse plant species Its remarkable adaptability allows it to flourish in challenging conditions, particularly in regions prone to deep flooding, where it establishes more readily than in shallow flooded areas.
The Dong Thap Muoi Center for Research Conservation and Development of Medicinal Plants, located in Binh Phong Thanh Command, Moc Hoa district, Long An Province, spans over 1,000 hectares of pristine Melaleuca forest, primarily featuring the medicinal M@ cajuputi This reserve also includes various international Melaleuca varieties, conserving 90 rare genetic resources, such as Desmodium styracifolium and Artemisia vulgaris Approximately 20 medicinal plant species are cultivated on nearly 100 hectares, adhering to Good Agricultural and Collection Practices (GACP) standards To support the development of medicinal herbs, the company has established the Moc Hoa Tram factory, compliant with GMP-WHO standards, which began production in late 2017 and has since launched over 30 natural product lines Collaborating with universities and institutions, the center focuses on creating optimal, non-genetically modified products while supplying clean raw materials to businesses nationwide.
The biodiversity situation in VI€tTaT -. 5 222221222 *+2E**ESEEErEerrrrerskrrrerrerree 8 2.7 The genelie IVETSILY - ccereeeneensenresnssansersereansenssansennennsennmacaucseneanternsanstansaaceeasens 9 2.8 Genetic markers and, their applications for genetic diversity analysis in plants LŨ 2:8, li Morpholo steal titat Mere csaysoe bi dong gi phửụgitgaSgsggiSDgiASbBSiSESISEgISÙSGBSQHHÄS:0g.h31BXSNgHA03/38.g83p 10 ÔN?) 09i xì l6
PCR-Based, molecular markers, Inter Simple Sequence Repeat (ISSR)
Polymerase chain reaction (PCR) is a rapid laboratory technique that amplifies millions to billions of DNA copies for detailed study It requires minimal DNA quantities, with simple extraction methods often providing sufficient quality PCR is quick and easy to perform once optimal conditions are established, and its versatile primer sequences enhance its diagnostic capabilities Common PCR-based techniques include Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), microsatellites or Simple Sequence Repeats (SSR), and Inter Simple Sequence Repeats (ISSR) (Idrees and Irshad, 2014).
Inter Simple Sequence Repeat (ISSR) is a PCR-based method used for amplifying DNA segments located between two identical microsatellite repeat regions that are oriented in opposite directions Introduced in 1994, ISSR markers have become a valuable tool in genetic research and analysis (Ng and Tan, 2015; Pradeep Reddy et al., 2002).
Microsatellite markers are widely utilized to assess genetic variation at the sub-species level, particularly in the study of population structure and differentiation in cultivated plants This technique employs inter-simple sequence repeat (ISSR) analysis, using short DNA sequences of 16 to 25 base pairs as primers in a single primer PCR reaction By targeting multiple genomic loci, ISSR amplifies inter-SSR sequences of various sizes The microsatellite primers can include di- or tri-nucleotide repeats, such as AC, GT, ACT, and GCA, with basic units repeated around ten times In plant genomes, other repeat types like GCTA (tetra-nucleotides) and GCATAC (penta-nucleotides) are also present The presence of a poly (GA) motif indicates a lower occurrence of the (GT) motif, while poly (AT) repeats, the most common di-nucleotides in plants, may lead to self-annealing issues that hinder amplification Conversely, primers based on (AG) and (GA) motifs have proven effective in generating distinct and clear amplification bands.
The polymorphism level in ISSR markers is influenced by the repeat motifs and the primer sequence used ISSR primers can be classified into three types: unanchored, 5’-anchored, and 3’-anchored Unanchored primers, which contain only repeated motifs, may cause amplification issues, leading to smears rather than distinct bands due to primer slippage Conversely, using anchored primers with additional nucleotides at the 5’ or 3’ end enhances specificity by ensuring that the primer anneals only to the ends of microsatellites, thus minimizing internal priming and smear formation.
Di-nucleotide repeats, commonly found at the 3' or 5' ends of DNA, demonstrate significant polymorphism (Joshi et al., 2000) These Simple Sequence Repeats (SSRs) are widely distributed throughout the genome, highlighting their potential for various genetic studies.
14 find two repeat sequences located adjacent to each other in the genome One can use such SSR sequences to generate ISSR markers.
Variety II, ISSR size: 18 bps ¢ c
Variety Ill, ISSR size: 15 bps
Figure 2.3 A hypothetical scheme of the concept of an ISSR marker defined by the intervening DNA sequence between the SSR,
CTCTCTCTCT (A), (B) and (C) show length polymorphism in three
Varieties I, II and III respectively using a single primer depicted as thick orange arrows (D) An electrophoretic gel depicting the band profile arising from variation in inter SSR nucleotides differences (hypothetically
23, 18 and 15 bps respectively) among the three Varieties I, II and III
ISSR markers are regarded as dominant markers in genetic studies, despite adhering to Mendelian inheritance patterns (Gupta et al., 1999) In Mendelian genetics, alleles at a locus can be dominant or recessive, with dominant alleles masking the expression of recessive ones The binary scoring system used for ISSR markers—where the presence of a band is marked as "1" and its absence as "0" (Wolfe and Liston, 1998)—does not differentiate between heterozygous and homozygous individuals A present band signifies a dominant allele, while its absence indicates either a recessive or null allele, reflecting potential sequence divergence at the primer's binding site.
Mutations, including deletions at specific loci or chromosomal rearrangements, can hinder DNA fragment amplification, resulting in polymorphism variations Additionally, if the amplified region fails to yield a product, it may lead to the absence of a detectable band (Wolfe and Liston, 1998).
ISSR markers demonstrate exceptional reproducibility, largely due to their longer primers (16 - 25 bp) compared to RAPD primers (10 bp), which enables higher annealing temperatures (45 - 60°C) and increased PCR stringency Research has shown that ISSR markers can achieve reproducibility levels exceeding 99%, as reported by Fang and Roose (1997), through various tests using DNA samples from the same cultivar across different locations, different-aged leaves from the same plant, and independent PCR runs Other studies have indicated that the reproducibility of ISSR amplification products can vary between 86% and 94% (Moreno et al., 1998).
ISSR markers combine the specificity of microsatellite markers with the advantage of not requiring sequence information for primer design, similar to random markers (Joshi et al., 2000) This technique presents multiple potential benefits for genetic analysis and research.
The ISSR technique offers several advantages, including simplicity and rapidity, minimal template DNA requirements, and small PCR reaction volumes, which reduce the need for large amounts of plant material for DNA extraction It allows for high variability in banding patterns and does not require specialized equipment, facilitating easy scoring of results Additionally, using higher annealing temperatures can minimize template-primer mismatch artifacts commonly seen with RAPD markers However, this method has limitations, such as the necessity for pure template DNA and standardized concentrations across samples, the need for initial reaction optimization, and the scoring of bands as dominant markers, which may affect genetic diversity estimates based on diallelic characters (Wolfe and Liston, 1998).
ISSR markers have been widely employed for the genetic characterization of numerous plant species Some of these are Myrcia lundiana Kiaersk (Alves et al,
2016), Myrcia ovata (Myrtaceae) (White et al., 2018), Oryza sativa L (Blair et al.,
Numerous studies, including those by Feitosa-Alcantara et al (2017) and Brito et al (2016), have employed ISSR markers for various applications These applications encompass genetic diversity analysis, fingerprinting, genome mapping, parentage determination, phylogenetic studies, gene tagging, and the identification of clones and strains Additionally, ISSR markers have been instrumental in taxonomic investigations of closely related species.
ISSR markers are preferred for genetic diversity analyses because they are simple, fast, cost-effective, and applicable to a wide range of plant species Their versatility is enhanced by the fact that they do not require genome sequence information for primer design, making ISSR primers accessible and affordable for researchers Consequently, this study selected ISSR markers due to these advantageous characteristics.
From September to December 2023, a study was conducted involving the collection of ten M cajuputi populations at the Dong Thap Muoi Center for Research Conservation and Development of Medicinal Plants.
Molecular analysis and evaluation of genetic distance were carried out at room
205 - Molecular Biology Department, Research Institute of Biotechnology and Environment - Nong Lam University Ho Chi Minh City.
3.2 Plant material and sampling strategy
In this study, ten populations of M cajuputi were collected from the Dong Thap Muoi Center for Research Conservation and Development of Medicinal Plants, with each population comprising five individuals Random sampling was conducted, gathering 2 to 3 branches from each tree, which included old, intermediate, and young leaves Each sample was placed in a labeled Zip bag, indicating the sample name and collection time, and stored in a Styrofoam box with gel ice to maintain leaf freshness during transportation Detailed lists of the ten M cajuputi populations utilized in this research are provided in Tables 3.1 and 3.2.
Table 3 1 List of ten ẤM cajuputi populations collected at the Dong Thap Muoi Center for Research Conservation and Development of Medicinal Plants
No Pop code Samples name Samples code GPS
Table 3 2.(continue) List of ten M cajuputi populations collected at the Dong Thap Muoi Center for Research Conservation and Development of Medicinal Plants
No Pop code Samples name Samples code GPS
3.3 Evaluation of genetic diversity of Melaleuca cajuputi populations based on morphological characteristics of the leaves.
In the study of M cajuputi populations, the procedures were followed for leaf data collection according to the International Plant Genetic Resources Institute standards (1999).
The collected samples were evaluated for their morphological characteristics in the laboratory, focusing on healthy, fresh branches The presence of fluff on the branch tips was noted, and photographs were taken to capture the leaf colors Measurements included the length from tip to base and the width at the widest point, with a selection of young, intermediate, and old leaves exhibiting sound morphology The analysis of leaf morphological characteristics was conducted using images and statistical data processed in Excel.
3.4 Using ISSR molecular markers to evaluate of genetic diversity of Melaleuca cajuputi populations.
The DNA extraction procedure followed the method outlined by (Doyle and Doyle, 1987):