MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESGRADUATION THESIS GENETIC DIVERSITY OF FIFTEEN DENDROBIUM SPECIES USING SRAP MARKERS..
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Dendrobium orchids are some off the most diverse orchids that exitst, diverse in shapes, colors, and sizes and is hence considered as a favoritet ornamental plant.
The Dendrobium genus, part of the Orchidaceae family, showcases diverse morphological traits and includes 98 species and one variety in Vietnam, categorized into 15 sections (Duong Duc Huyen, 1992) Distinct from neighboring genera, Dendrobium species typically grow in clusters, feature segmented structures, possess four pollen masses, and lack soft hairs on their leaves and floral parts.
Dendrobium species have up to 100 species, divided into 14 tribes, can be identified by their pseudobulbs, leaves and flowers (Tran Hop, 1998).
The tree features an aerial root system enveloped in a thick, hygroscopic layer of dead cells filled with air, giving the roots a silvery appearance This unique structure allows the roots to absorb rainwater as it flows down the bark or from moisture in the air, facilitating the uptake of essential nutrients and minerals Additionally, the roots anchor the tree securely to the substrate, which can limit air circulation Some species exhibit underdeveloped stems and leaves, relying on chlorophyll-rich root systems to capture light for photosynthesis and flowering Notably, the roots of dendrobium orchids are sensitive to cold temperatures (Nguyen Cong Nghiep, 2000).
If the roots are subjected to prolonged cold temperatures, they will deteriorate and ultimately lead to the demise of the plant (Duong Cong Kien, 2006).
Dendrobium orchids, classified as sympodial orchids, feature unique structures known as pseudobulbs, which serve both as roots and nutrient storage These thickened segments, filled with a gel-like substance, help minimize water loss and store essential nutrients, enabling the plant to thrive in dry conditions Pseudobulbs, which can vary in shape from spherical to elongated, also contain chlorophyll for photosynthesis In colder climates, some pseudobulbs may lack the typical green coloration and primarily function as nutrient reservoirs while still supporting leaves at the top.
Most dendrobium species are autotrophic, featuring a fully developed leaf system Their leaves are simple and undivided, exhibiting a wide range of shapes, from succulent to narrow.
The foliage displays a needle-like shape with either grooved or tapered leaves These leaves have a soft, fleshy texture and a glossy green color that varies in intensity based on environmental conditions The leaf blades can also broaden significantly.
Leaves may exhibit pleating along the arcuate veins, resembling a fan, or they can fold unilaterally along the midrib in a 'V' shape In some cases, basal leaves may reduce to vestigial structures or scale-like formations (Duong Cong Kien, 2006).
Tropical dendrobium orchids, part of the Orchidaceae family, typically shed their leaves during the dry season After this period, the plants may either bloom or enter dormancy until the onset of the rainy season stimulates the emergence of new buds (Tran Van Bao, 2009).
In species with persistent leaves, flowers are arranged in charming pairs, threes, or closely spaced clusters, creating elegant upright or hanging formations Conversely, another group features small, solitary flowers that gracefully emerge from the leaf axils, showcasing a remarkable diversity in dimensions, sizes, and vibrant colors at the tips or near the tips of the plants.
Dendrobium orchids are celebrated for their stunning beauty, making them a popular choice as cut flowers that elevate indoor decor These versatile plants also thrive as potted specimens or in hanging baskets, effortlessly adding charm and elegance to any environment.
The Orchidaceae family produces capsule-shaped fruits that burst open when seeds mature, with the capsules remaining attached only at the top and bottom In certain species, the fruit stays closed even after ripening, releasing seeds only upon decomposition.
Fruits can contain a significant number of seeds, varying from 10,000 to 100,000, and in some cases, reaching up to 3 million These tiny seeds have undifferentiated embryos and take about 3 to 5 months to mature before being dispersed by the wind (Duong Cong Kien, 2006).
Seeds lacking nutrients are scattered by the wind To initiate germination, these seeds rely on symbiotic fungi to provide necessary substances, especially during the initial stages of development.
Genetic diversity refers to the range of hereditary traits within a species, arising from the recombination of genetic material during inheritance This diversity fluctuates over time and across different locations Species with high genetic diversity usually consist of many individuals showcasing a variety of characteristics The ability of a population to adapt to changing environments largely depends on its level of genetic diversity.
Genetic variation within a species is driven by multiple factors, including mutations that change gene sequences and contribute to diversity Another key source is gene flow, which involves the transfer of genes between different populations Furthermore, sexual reproduction creates new combinations of genes, further enriching genetic variation.
Genetic diversity plays a crucial role in determining physical traits and the ability to adapt to stress, illness, and challenging environments Natural selection, influenced by both natural and human-induced environmental changes, tends to favor individuals with beneficial traits As a result, populations with higher genetic diversity are more likely to thrive, while those with lower adaptability may face extinction.
Maintaining genetic diversity is essential for community health, as it fosters the development of plant varieties resistant to pests and diseases By crossbreeding different genetic variants, we can create new plants with beneficial traits such as disease resistance and stress tolerance Additionally, genetic diversity minimizes the risk of undesirable inherited traits and enhances the survival chances of species.
2.2.2 The signficance of genetic diversity
Genetic diversity plays a crucial role in ensuring the long-term reproductive success, resilience, and ability to adapt of all species, as they navigate through changing environmental circumstances.
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Genetic diversity analysis among dendrobium SpeC1€S ~ <+s 25 4.1.4 Genetic distance and cluster analysis of 15 dendrobium specl1es
Regarding the leverls of genetic diversity among 15 dendrobium species in this sity, dendrobium species in the study show high heterozygosity values resulting in high diversity.
Statistical analysis of various species reveals key genetic metrics, with the average observed number of alleles (Na) at 1.746, the effective number of alleles (Ne) at 1.307, Nei’s gene diversity (H) at 0.190, and Shannon’s information index (I) averaging 0.402 Notably, Shannon’s index varies across samples, ranging from a low of 0.140 (S10) to a high of 0.504 (S2).
According to the allele distribution statistics in Figure 4.2, all reproducible polymorphic loci exhibited frequencies exceeding 5% The TQTD population showed the highest number of bands at 179, while the GYD population had the lowest with 159 Additionally, the occurrence of less common bands was noted in 25% and 50% of the populations.
Table 4.3 Genetic diversity parameters of dendrobium species analyzed it Abbr N Na Ne I H
Na: No of Different Alleles; Ne: No of Effective Alleles); I: Shannon's
Information Index; H: Nei’s gene diversity (1973)
Genetic diversity is quantified by the observed number of alleles, which indicates the variety of gene forms present in a population Typically, populations with greater genetic diversity showcase a higher count of different alleles, reflecting their rich genetic variation.
A diverse array of allele variations plays a crucial role in improving population adaptability to environmental changes and minimizing inbreeding risks The average number of alleles identified in each sample is approximately 1.746, which is notably lower than the findings of Tran Thi Thuy Ha and colleagues (2019), who reported a higher number of alleles (Na).
In a study conducted by Bui Ha My and colleagues (2018), the Na levels were observed to range from 2 to 4 for the age group of 6 to 18 However, due to the restricted distribution area and the small sample size, it is justifiable to consider a lower Na value.
The effective number of alleles (Ne) serves as a crucial indicator of genetic diversity, reflecting both the quantity and frequency of alleles within a population In this study, Ne values range from 1.209 to 1.421, averaging at 1.307, which suggests a relatively balanced genetic diversity among individuals Although these Ne values are lower compared to findings by Tran Thi Thuy Ha et al (2019) and Bui Ha My et al (2018), they are influenced by factors such as sample size, allele count, and the population's genetic traits.
The study's limited sample size suggests a potential bottleneck phenomenon in the dendrobium population, likely due to population dispersal or a sudden decrease in effective population size This scenario increases the risk of inbreeding and uneven genetic drift, leading to a reduced number of alleles The findings highlight a decline in the natural dendrobium population, underscoring the urgent need for effective conservation measures.
Nei’s gene diversity (H) takes into account both the quantity and frequency of alleles at a specific location A higher Nei’s gene diversity value indicates greater genetic diversity.
This study assessed Nei's gene diversity values, which varied from 0.099 to 0.259, averaging at 0.190, indicating a low level of genetic diversity among the samples Additionally, the Shannon's diversity index ranged from 0.140 to 0.504, with an average of 0.402, further supporting the observation of limited genetic variation.
4.1.4 Genetic distance and cluster analysis of 15 dendrobium species
A matrix simple matching analysis of 15 dendrobium species shows significant genetic similarities among them, as detailed in Appendix 1 Notably, the pairs S11 and S10, as well as S13 and S14, exhibit the highest genetic similarity coefficient of 0.827, indicating their close relationship Conversely, the comparison between S6 and S14 reveals the most considerable genetic distance, with a similarity coefficient of 0.628.
The results of the genetic relationship analysis and the UPGMA analysis, shown in Figure 4.5, the dendrogram indicated that five main clades were designated as “I”, “II”,
“II”, “IV” and “V” of which cluster III contained two clades as C and D Cluster C was characterized by seven species including S5, S9, S12, S13, S14, S10, S11 and S15.
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Figure 4.5 Dendrogram generated from UPGMA clustering method using Simple Matching coefficients of 15 dendrobium species based on SRAP analysis.
The effectiveness of using the SRAP marker in genetic dIVerSIty - 29 4.2.2 Genetic diversity among 15 dendrobium species :.ccecceceeceeseeseseeteeteeeeseeseens 29 4.2.3 Genetic relationships of 15 dendrobium species .cceccssceeceeeceeeeeeeeseeteeeseeeees 30 CHAPTER 5 CONCLUSION AND SUGGESTION 00 0 cccscssscssseceseesesecsseeeseeseeneoes 31 REFERENCES saceeaaaaadeeiiuiiiobibodietoosbobiEESL101358058DA421615SELISS42383383898383355SG:XBESE4E.2 SE08 32
In a study involving 30 primers, 16 were found to exhibit polymorphic segments, highlighting their importance in assessing the genetic diversity of dendrobium species The presence of these polymorphic segments is quantified using metrics like PIC, which underscores their role in genetic analysis.
In a comprehensive study assessing the genetic diversity of 15 Dendrobium species, various markers were utilized, with the Polymorphism Information Content (PIC) serving as a crucial indicator of genetic variation A PIC value exceeding 0.5 indicates a high level of polymorphism, while values between 0.5 and 0.25 suggest medium polymorphism, and values below 0.25 indicate low polymorphism (Botstein et al., 1980) The findings revealed that SRAP markers exhibited a significantly higher average PIC value of 0.891, indicating high polymorphism, compared to SCoT markers, which had a PIC value of 0.43 Overall, SRAP markers demonstrated a PIC range from 0.18 to 0.99, reinforcing their effectiveness in assessing genetic diversity (Paromik Bhattacharyya and Suman Kumaria, 2013).
The findings in Table 4.2 reveal that the SRAP marker demonstrates a significant level of amplification throughout the dendrobium genome, as indicated by the high number of successfully amplified bands In the study by Paromik Bhattacharyya (2013), Scot markers yielded 127 bands from 16 primers, averaging 8.25 bands per primer, but only showed a low percentage of polymorphic fragments at 56.82% In comparison, the SRAP marker achieved an average of approximately 8.874 bands per primer, with a notably higher percentage of polymorphic fragments at 76.95%.
Hence, the comparison of PIC values indicates that the SRAP marker is one of the potential markers for assessing genetic diversity characteristics and genetic differences applicable to dendrobium species.
4.2.2 Genetic diversity among 15 dendrobium species
An analysis of the genetic diversity among 15 dendrobium species from the orchid collection of Professor Bui Cach Tuyen reveals an average to high level of diversity, with a diversity index of 0.402.
The analysis reveals that sample S1 exhibits the highest diversity with an index of 0.504, while S10 shows the lowest diversity at 0.120 Correspondingly, the Shannon diversity index aligns with the genetic diversity coefficient (H), which ranges from 0.099 to 0.259 Notably, S7 possesses the highest genetic diversity coefficient at 0.259, whereas S1 has the lowest at 0.099, falling below the average coefficient of 0.190 These findings suggest that S1's unique genetic diversity, compared to other samples from the same region and species, is likely influenced by hybridization and the area's natural conditions.
4.2.3 Genetic relationships of 15 dendrobium species
An analysis of the genetic relationships among 15 dendrobium species, utilizing UPGMA hierarchical clustering, demonstrates high genetic similarity coefficients ranging from 0.628 to 0.827 At a genetic similarity threshold of 0.724, these species are categorized into five distinct clusters, labeled Clade I, II, IIL, IV, and V.
Clade I included 3 samples is S1, S2 and S3 Clade III included 9 samples is S5, S9, S12, S13, S14, S10, S11, S15 and S8 The other 3 clades have only 1 sample for each.
Clade I include 3 samples: S2, S2 and S3 have the numorous similar hereditary traits.
7 dendrobium species clasified in cluster B included S5, S9, S12, S13, S14, S10, S11, S15 and S8 in cluster C, have closed relationship.
Clade III consists 9 samples, S10 and S11 exhibit a highest genetic similarity coefficient of 0.827, S6 and S8 have a lowest genetic similarity.
This study is the first to utilize SRAP markers for assessing the genetic diversity of dendrobium species The findings demonstrate that SRAP markers are effective for exploring genetic diversity within extensive collections, enabling the differentiation between native and non-native dendrobium species in Vietnam.
This study highlights significant genetic diversity among 15 dendrobium species, with SRAP primers yielding a notable number of polymorphic markers Out of 30 tested primer pairs, 16 were found to be effective and significant in assessing the genetic relationships among these species, demonstrating varying rates of polymorphism.
57 to 100% with an average PIC polymorphic information content value of 0.891 per markers from the dendrobium genome.
The pairwise genetic similarity among the 15 dendrobium species studied ranged from 0.724 to 0.827, with genetic categorization performed using the UPGMA method and NTSYSpc 2.1 program, revealing a single category for dendrobium Significant genetic similarities were observed in fruit quantity, leaves, roots, and flowers within the same group The findings indicate that all species exhibit a strong original correlation and possess numerous alleles in their genomes related to various traits and morphology This research enhances the understanding of genetic diversity among dendrobium species in Vietnam and provides valuable insights for breeding programs.
To enhance the understanding of genetic diversity among Dendrobium species in Vietnam, it is essential to continue evaluating this diversity through the use of additional molecular markers This approach will strengthen the reliability of the data collected.
Towards constructing effective molecular markers, construction of novel markers for microsatellite areas and candidate genes for successful dendrobium selection and breeding.
Dương Đức Huyền (1992) đã thực hiện nghiên cứu phân loại chi Hoàng thao - Dendrobium Sw thuộc họ Lan Orchidaceae ở Việt Nam Luận án phó tiến sĩ của ông được bảo vệ tại Viện Khoa học Việt Nam, đóng góp quan trọng vào lĩnh vực sinh học và nghiên cứu thực vật.
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Appendix 1: Dendrogram generated from UPGMA clustering method using Simple Matching coefficients of 15 dendrobium species based on SRAP primer
Appendix 2: The electrophoresis results on a 1.5% agarose gel using the SRAP technique with 16 primers showed successful amplification
Fig 1 Mel - Em3 PCR amplification.
Figure 3 Me2 - Em1 PCR amplification.
Figure 5 Me2 - Em6 PCR amplification.
Figure 7 Me3 - Em2 PCR amplification.
Figure 9 Me3 - Em6 PCR amplification.
Figure 12 Me5 - Em2 PCR amplification
Figure 13 Me5 - Em3 PCR amplification.