1 INTRODUCTION 1 The importance of the topic Plant genetic resources play an important role in agricultural production towards diversification and modernization In the sustainable exploitation and use[.]
1 INTRODUCTION The importance of the topic Plant genetic resources play an important role in agricultural production towards diversification and modernization In the sustainable exploitation and use of plant genetic resources, the assessment of genotyping is an extremely crucial step not only for identifying and distinguishing between different varieties/species but also for understanding their genetic relationships The significant development of molecular biology methods and techniques has created various effective applied tools in the study of diversity in terms of genotype Currently, the method of using molecular markers in genetic identification research has been applied intensively Using the DNA barcode marker gene region is a new tool, which is being widely applied in the world, based on the evolution and nucleotide sequence difference, allowing classification and identification of genetics relationships between different varieties/species Nelumbo nucifera Gaertn (N nucifera) is a species of lotus belonging to the group of perennial aquatic plants living in ponds and lakes, grown in many parts of the world In Thua Thien Hue, there are several varieties of lotus with different flower colors being grown in various places including suburban and inner city areas The Concave white lotus is an ancient native of Hue’s lotus variety, known as a famous royal lotus variety Tourists are attracted by this lotus variety because it has a gentle fragrance, white flowers, lotus roots and seeds with special taste and quality In recent years, the lotus growing in Hue has been invested and developed strongly Some localities have converted inefficient rice growing areas to lotus areas in order to commercial lotus seeds Therefore, in addition to planting white lotus varieties of Hue origin, people have also imported the high yield varieties from different localities to grow in Hue, especially the high-yield pink lotus variety This species is specialized for seeds and has outstanding characteristics in terms of living conditions and yields than Hue’s lotus varieties The cultivation of many types of imported lotus species in Hue has led to the confusion and degeneration of Hue’s lotus varieties and then reduced to the number of indigenous lotus varieties in Hue The lotus plant has been widely used in many areas of life such as decorative arts, ornamental flowers, spiritual culture, cuisine, medicine, and so on Many current studies have showed that the different parts of lotus plants can be used as a food or valuable traditional medicine because it contains many compounds of the nature of alkaloids and flavonoids which have positive effect on human health such as anti-inflammatory, antiamnesia, anti-oxidant and tumor suppressor activity Therefore, the study of genetic differences between lotus species grown in Hue by using genetic markers is necessary to identify the genetic differences in specific genotypes to assess the biological activity of lotus seeds Starting from the above scientific basis, I carry out the topic: “The study difference in nucleotide sequences of the marker gene regions in lotus varieties grown in Hue and investigate the biological activities of some extracted compounds from lotus seeds” Hence, this study will provide a database on the molecular level of some marker gene regions, establish genetic relationships in different lotus species grown in Hue, and provide scientific data on the biological activities of some compounds extracted from local lotus seeds Objectives 2.1 General objective Studying genetic differences between lotus varieties grown in Hue based on some gene markers and investigating biological activities of compounds extracted from local lotus seeds 2.2 Specific objectives - Provide the scientific data of the nucleotide sequence difference on the marker gene regions obtained from lotus samples grown at different locations in Hue, deposit data and receive codes on GenBank 3 - Evaluate the application of marker gene regions of nuclear genome and chloroplast genome to detect differences in nucleotide sequences of gene regions obtained from lotus samples grown in different locations in Hue Investigate the rules of differences, establish phylogenetic relationships based on gene regions that have nucleotide differences between some lotus samples grown in Hue, serving as a scientific basis for the selection of distinctively different varieties to study of bioactive compounds - Research on isolating purified compounds and testing biological activities of compounds extracted from lotus seeds of selected lotus varieties, creating a premise for the sustainable exploitation and use of lotus seeds in Hue Noval contributions - The project has contributed new results as follows: Constructed a data set of nucleotide sequence differences of marker gene region from the nuclear genome and marker gene regions from the chloroplast genome of 33 lotus samples grown at different locations in Hue Since then, the result has deposited and been granted access codes for 330 nucleotide sequences of 10 marker gene regions obtained from 33 lotus samples in Hue on GenBank Initially discovered the rule of the difference in nucleotide sequences of some marker gene regions between 33 lotus samples grown at different locations in Hue, building a phylogenetic tree between 33 lotus samples based on the nucleotide differences in the indicator gene regions, and selection of the Concave white lotus variety with specific differences in nucleotide sequences to study biologically active compounds in lotus seeds Initially, three pure compounds were isolated from the n-Butanol fraction of Concave white lotus seeds with alkaloid nature: nuciferine (C19H21NO2), armepavine (C19H23O3N), and anonaine (C17H15NO2) Initially, these three compounds had strong antioxidant activity but did not show cytotoxic activity against three cancer cell lines MKN7, SKMel-2 and KB at all the treatment concentrations 4 The structure of the thesis The thesis consists of 139 pages Introduction is 05 pages, conclusion and recommendations of 02 pages, published works of 01 page, 23 pages for references and appendices The main content of the thesis is divided into 03 chapters: Chapter 1: Literature Review consists of 44 pages; Chapter 2: Materials and Research Methods is 18 pages; and Chapter 3: Results and discussion 46 pages CHAPTER LITERATURE REVIEW The literature review gathers the latest domestic and international studies on the application of molecular markers in genetic diversity analysis, and species identification in plants in general and in the species lotus (N nucifera) in particular Studies on extracting compounds from different parts of the lotus plant and testing their biological activities CHAPTER MATERIALS AND RESEARCH METHODS 2.1 Materials and subjects of study 2.1.1 Materials lotus Materials used in genetic research: Leaves of 33 lotus samples were collected at 33 different lotus growing sites in Hue Lotus samples were collected based on published results of Nguyen Thi Quynh Trang et al (2017) based on morphological characteristics of a group of 66 lotus samples collected in Hue Materials used for compound extraction: Seeds of the Concave white lotus (ST02) collected at Tinh Tam lake, Thuan Loc ward, Hue city 2.1.2 Materials used in molecular biology: The ten pairs of barcode primers specific for 10 DNA marker gene regions were referenced from Dong et al (2012), Cuenoud et al (2002), and White et al (1990) 2.2 Research Methods 2.2.1 Molecular biology method 2.2.1.1 Chloroplast extraction: Lotus leaves are stored at 4°C in the dark for about to days to remove part of the starch present in leaf tissue Chloroplasts in lotus leaves were extracted and purified as described by Lang et al (2011) 5 2.2.1.2 Isolation of DNA from nuclear genome and cpDNA from chloroplast: Lotus leaves and chloroplasts were used as raw materials for the extraction and purification of DNA and cpDNA based on the CTAB method as described by Doyle (1987) 2.2.1.3 Method for determining the content and purity of DNA and cpDNA: The quality of DNA and cpDNA will be checked on agarose gel electrophoresis and spectrophotometric methods on Nanodrop ND1000 (Thermo) 2.2.1.4 The PCR reaction: PCR amplification was performed on thermal cycler (MJ-MiniTM Persanol Thermal Cycle, BioRad) according to the following cycling conditions: 95oC/5 minutes; followed by 30 cycles: 95oC/1 minute, 50 - 55oC/50 seconds, 72oC/1 minute, and 72oC/10 minutes 2.2.1.5 Method for the purification of PCR products: PCR products are purified by Isolate II PCR and Gel Kit (Bioline) 2.3.1.6 Gene cloning method: Method TA cloning into the vector pGEM® -T Easy was used to clone the genes 2.2.1.7 Plasmid purification methods: Recombinant plasmid DNA was purified from positive colony lines using the EZ-10 Spin Column Plasmid DNA Minipreps kit, BS6141 (BioBase) 2.2.1.8 Sequencing method: Target genes were sequenced based on Sanger fluorescent marker method on the ABI 3100 Avant Genetic Analyzer system 2.2.1.9 Genetic analysis: Nucleotide sequences were sorted using the CLUSTALS program and corrected using BioEdit 7.0.5 software The Phylogenetic tree was built using MEGA X software on the Maximum Like hood (ML) method The nucleotide sequences of the gene regions were compared with the nucleotide sequences published on GeneBank using the BLAST program The DNA polymorphism analysis based on eight parameters including number of separate polymorphic sites (S), total number of mutant sites (Eta), number of haplotypes (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity () minimum number of recombinant processes (Rm) and number of effective populations for mutation rate at each nucleotide position per generation (Ø) are considered as a polymorphic measurement in the population Neutrality is tested based on five methods namely (Tajima’s D test (Tajima, 1989), Fs, Fu’s statistic (Fu, 1997); S, Strobeck’s statistic (Stroreck, 1987); D* and F*, Fu and Li’s statistics (Fu and Li, 1993) were used to DNAsp 6.0 software (Rozas et al 2017) Population segregation statistics (FST) were estimated using the method of Hudson et al (1992) 2.2.2 The methods of extraction of compounds and biological activity testing 2.2.2.1 Method of segment extracts collection: Sample treatment, sample extraction with 70% ethanol, cold-soaking in an acid environment, fractional extraction with polar solvents ethyl acetate, n-Butanol From the n-Bu fraction of Concave white lotus seeds, the active ingredients were extracted and isolated by methods of thin-layer chromatography (TLC), ordinary column chromatography (CC) with the stationary phase of silica gel (Merck) 2.2.2.2 Method for determination of purity and structure of compounds a Thin layer chromatography Method: Thin layer chromatography of the isolated compounds was performed as described by Bela and Khale (2011) b HPLC-MS/MS spectral analysis method: Analyze MS/MS mass spectra on the Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer system The MS/MS spectral data of the active ingredients were looked up and compared by us with standard spectral data on the NIST2017 spectrum library and related publications 2.2.2.3 The test method for biological activity a The method of determining the antioxidant activity of the crude extract and the purified active compounds was carried out by the 1,1-diphenyl-2picrylhydrazyl (DPPH) method as described by Vuong et al (2013) b In vitro cancer cell line culture: Cancer cell lines were cultured as a monolayer in DMEM culture medium by adding 10% fetal bovine serumFBS Cells were transplanted after 3-5 days at a ratio (1:3) and kept in incubators at 37C, 5% CO2 c Determination of cancer cytotoxicity against monolayer cultured cells: The assay was carried out to determine the total protein content based on the optical density measured when the protein composition of cells was stained with Sulforhodamine B (SRB, Sigma, USA) d Bioassay for cytotoxicity against cell lines of suspension cultures (HL60): Method using tetrazolium salts as a reagent in the colorimetric assay, thereby assessing cell survival and viability 2.2.3 Data analysis methods: Experimental experiments were repeated times The analytical values were statistically significant with p < 0.05, the analytical values were statistically significant Data processing using SPSS software was used for all statistical analyses (Version 20) CHAPTER RESULTS AND DISCUSSIO 3.1 The PCR amplification results and analysis of nucleotide sequences of some marker gene regions The results of PCR amplification products of 10 marker gene regions shown on the electrophoresis in Figure 3.1 show that all lotus samples used in the study gave 100% PCR amplification rate, clear DNA bands, high concentration, quality assurance as raw materials for subsequent experiments (Figure 3.1) A B C D E F G H I K Figure 3.1 The electrophoresis of PCR products M1: DNA standard ladder, Figure A-K are PCR results of ITS4-5, rbcL, matK, trnH -psbA, accD-psaI, ndhA, psbE-petL, Rpl32-trnL, trnW-psaJ and trnSGCU-trnGGCC gene regions, respectively; NC: Result of negative control PCR product; wells from 01 to 11 PCR product results of the white lotus samples; wells from 12 to 33 PCR product results of pink lotus samples 8 The size of 10 marker gene regions collected for 33 lotus samples ranged from 350 bp to 1667 bp In which the ITS4-5 and trnH-psbA gene regions obtained nucleotide sizes that differed between lotus samples, ranging from 729 bp to 744 bp (ITS4-5) and from 350 to 410 bp (trnH-psbA) (Table 3.1) The nucleotide sequence of each DNA marker gene region collected from 33 lotus samples compared with the nucleotide sequence published in GenBank showed high similarity with lotus species N nucifera (Code: FJ599761.1 and KF009944.1), similarity levels ranged from 96.89-100% The nucleotide sequences of 10 DNA marker gene regions were collected from 33 lotus samples deposited in GenBank with reference codes ITS4-5 (MT903421MT903453), rbcL (MN011708-MN068956), matK (MN011719- MN068978), trnH-psbA (MN011730-MN086252), accD-psaI (MN086253-MN086285), psbE-petL (MT901764-MT901796), Rpl32-trnL (MT901731-MT901763), trnW-psaJ (MT905225-SGCU-5257), trnSGCU-trnGGCC (MT905258 to MT905290) and ndhA (MZ611976 -MZ612008) 3.2 The results of genetic characteristic analysis of some lotus samples grown in Hue based on 10 gene markers 3.2.1 The results of molecular characterization of 10 marker gene regions obtained from some lotus samples grown in Hue The ITS4-5 gene region collected from 33 different lotus samples showed that Cysteine (C) accounted for the highest percentage (mean = 30.538%) and the lowest was Timin (Uracin) (mean = 20,521%), the % (G+C) between the lotus samples averaged 55.111% The chloroplast marker gene regions, type A nucleotides accounted for the highest proportion for rbcL, trnH-psbA, and psbE-petL gene regions, with an average of 28.802%, 45.980%, and 33.413%, respectively Timin (Uracil) accounted for the highest proportion for the remaining gene regions such as matK, accD-psaI, ndhA, Rpl32-trnL, trnW-psaJ, trnSGCU-trnGGCC, the average obtained was 34.612%, 34.724%, 34.922%, 35.072%, 33.396%, and 35.137%, respectively The % (G+C) in each chloroplast marker gene region averaged from 24.121% (trnH-psbA) to 43.476% (rbcL) and this rate for accD-psaI, ndhA, psbE-petL, and Rpl32-trnL did not differ between lotus samples (Table 3.1) 3.2.2 The results of the analysis of genetic characteristics of 10 marker gene regions obtained from some lotus samples grown in Hue Table 3.1 Results of analysis of molecular and genetic characteristics of 10 marker gene regions obtained from some lotus samples grown in Hue Gene Size Number of S Eta Variable region (bp) monomorphic sites sites (%) ITS4-5 729-744 724 5 2,688 rbcL 743 742 1 0,135 matK 936 861 75 76 8,013 trnH-psbA 350-410 375 0 accD-psaI 887 887 0 ndhA 1206 1206 0 psbE-petL 1667 1667 0 Rpl32-trnL 1246 1239 7 0,562 trnW-psaJ 729 728 1 0,137 trnSGCU1097 1095 1 0,182 trnGGCC Note: S: Number of variable sites; Eta: Total number of mutations Medium % (G+C ) 55,111 43,476 36,000 24,121 30,800 33,900 33,700 30,000 34,200 31,400 The results of nucleotide sequence analysis showed that there were five polymorphic (S) nucleotide positions found in the ITS4-5 gene region between 33 lotus samples, accounting for 2.688% and one loss nucleotide sequence from positions 133 to 147 consisting of 15 nucleotides ACGTCCAGCATTCCA (Figure 3.2 and Table 3.1) The average number of different nucleotides k = 2,292, corresponding is π = 3.140 x10 -3 Figure 3.2 The results of analysis and comparison of nucleotide sequences in the ITS4-5 marker gene region among some lotus samples 10 The gene regions of the chloroplast genome have a variable number of nucleotide positions in the total length of the gene region obtained in 33 lotus samples, ranging from to 75 positions, corresponding to the percentage of polymorphic nucleotides that appeared on nucleotide sequences in gene regions ranging from to 8.013% (Table 3.1) The matK gene region has the largest number of variable nucleotide positions (75 positions), of which there are 74 polymorphic nucleotide positions, accounting for 8.013% (Table 3.1 and Figure 3.3) The results of genetic analysis based on parameters k, π, S, Eta among 33 lotus samples in each chloroplast gene region were different, ranging from 0.061 to 20.563 (k), 0.080 x10-³ to 21.970 x10-³ (π), to 75 (S) and to 76 (Eta) The rbcL gene region showed that these parameters obtained the lowest values (k = 0.061 and π = 0.080 x10-³, S = and Eta = 1), followed by the trnH-psbA gene regions (k = 0.117 and π = 0.330 x10-³), Rpl32-trnL (k = 0.424 and π = 0.340 x10-³), trnSGCU-trnGGCC (k = 0.504 and π = 0.460 x10-³), trnWpsaJ (k = 0.511 and π = 0,700 x10-³) and matK gene region, these values were obtained the highest (k = 20.563 and π = 21.970 x10-³) (Table 3.1 and 3.2) Figure 3.3 The results of analysis and comparison of nucleotide sequences in the matK marker gene region among some lotus samples Lotus samples are divided into haplotypes has differences in each region gene marker, ranging from to haplotypes, corresponding to the obtained Hd ranging from to 0.822 In which the matK gene region, these values 11 were obtained, respectively, at h = 5, and Hd = 0.822 (Table 3.2) The chloroplast marker gene regions such as rbcL, trnW-psaJ, and trnSGCUtrnGGCC contain to variable nucleotide positions in the genomic sequence The percentages of polymorphic nucleotides of the gene regions are 0.135%, 0.137%, and 0.182%, respectively (Table 3.1) Table 3.2 The Results of the analysis of genetic characteristics of 10 gene regions and combinations of marker gene regions Gene region ITS4-5 rbcL matK trnH-psbA accD-psaI ndhA psbE-petL Rpl32-trnL trnW-psaJ trnSGCU-trnGGCC ITS4-5 + rbcL ITS4-5 + matK ITS4-5 + Rpl32-trnL ITS4-5 + trnW-psaJ ITS4-5 + trnSGCU-trnGGCC rbcL + matK + Rpl32-trnL + trnW-psaJ + trnSGCU-trnGGCC A B π (x10-3) 3,140 0,080 21,970 0,330 0 0,340 0,700 0,460 1,600 13,730 1,380 1,920 1,530 k 2,292 0,061 20,563 0,117 0,424 0,511 0,504 2,352 22,854 2,716 2,803 2,795 Rm 0 0 0 0 0 1 h 2 1 2 4 Hd 0,458 0,061 0,822 0,117 0 0,061 0,511 0,504 0,477 0,845 0,498 0,689 0,699 4,640 22,063 0,869 C D Figure 3.4 The results of analysis and comparison of nucleotide sequences of marker gene regions between some lotus samples grown in Hue A: gene regions rbcL, B: gene regions Rpl32-trnL, C: gene regions trnW-psaJ D: gene regions trnSGCU-trnGGCC 12 The sample high-yield lotus SH09 has different nucleotide positions in the Rpl32-trnL gene region sequence compared to other lotus samples The percentage of polymorphic nucleotide positions is 0.562% (Figure 3.4B and Table 3.1) The Rpl32-trnL gene region with mutant sites was formed with the distribution of lotus samples in haplotypes being different, appearing that one haplotype contained only one sample of sample highyield lotus SH09 (Table 3.1) The results of nucleotide differences in the sequences of each gene region rbcL, trnW-psaJ, and trnSGCU-trnGGCC caused the lotus samples with different flower colors to division about into unclear haplotypes Which, the rbcL gene region appeared haplotype with a difference, appearing that one haplotype containing only one of the sample Concave white lotus ST02 (Figure 3.4) The trnH-psbA gene region with the TAAAA repeat sequence was different between the lotus samples from position 242 to 300, ranging from to 19 times In, the sample Concave white lotus ST02 has 13 repeats of the TAAAA sequence in the gene region This number of repetitions did not appear in other the sample lotus used in the study (Figure 3.5) The difference in the number of ATAAA repeats in the trnH-psbA gene region contributes to the division of 33 lotus samples into different haplotypes, and appearing that one haplotype containing only two lotus samples belonging to the high-yield lotus group SH02 and SH13 (Table 3.2) The accD-psaI, ndhA, and psbE-petL gene regions showed no nucleotide differences in the gene regions between the lotus samples used in the study Figure 3.5 The results of analysis and comparison of nucleotide sequences in the trnH-psbA marker gene region some lotus samples 13 The analysis of genetic characteristics (k, S, Eta) of 33 lotus samples is based on the combined analysis of the nucleotide sequences of the ITS4-5 gene region and the nucleotide sequences of the chloroplast marker gene regions nucleotide difference between 33 lotus samples The results showed a different variation compared to the results obtained when analyzing each individual gene region, ranging from 2.352 to 22.854 (k), 1.380 x10 -3 to 13.730 x10-3 (π), to 85 (S), to 86 (Eta) (Table 3.1 and 3.2) The results of the distribution of lotus samples in terms of haplotypes were different, many new haplotypes appeared and dominated the number of studied lotus samples, ranging from to haplotypes and the coefficient form of haplotypes ranges from 0.477 to 0.869 (Table 3.2) Gene region combinations ITS4-5 + rbcL, ITS4-5 + trnW-psaJ, ITS4-5 + trnSGCUtrnGGCC, ITS4-5 + Rpl32-trnL, and rbcL + matK + Rpl32-trnL + trnW-psaJ + trnSGCU-trnGGCC appearing one haplotype containing only one specimen of sample Concave white lotus ST02 or High-yield-pink SH06 or SH09 The minimum number of recombination events (Rm) was not found in the marker gene regions among 33 lotus samples This event only occurred in a few gene region combinations such as ITS4-5 + trnW-psaJ, ITS4-5 + trnSGCU-trnGGCC, rbcL + matK + Rpl32-trnL + trnW-psaJ + trnSGCU-trnGGCC (Table 3.2) Summary The analysis results showed that gene markers out of 10 gene regions used in the study had nucleotide differences between 33 lotus samples grown at different locations in Hue The matK gene region gave the largest number of nucleotide positions difference between the 33 lotus samples, followed by the ITS4-5 gene region, the Rpl32-trnL gene region, and the lowest rbcL gene region The gene regions showing the difference according to a general rule among lotus samples expressed through the respective nucleotide positions are ITS4-5: 404 (C-T), 625 (G-A) and 626 (A-G), 408 (G-T) ) and 576 (C-A); rbcL: 459 (G-T); trnW-psaJ: 504 (T-G); trnSGCU-trnGGCC: 369 (A0) and 545 (A-T); Rpl32-trnL: 885 (C-G), 895 (CATC-ATCA), 920 (A-G) and 927 (A-G); matK: 293 (G-A), 323 (G-A-T), 709 (G-T) and 819 (T-G) and the number of ATAAA repeats of the trnH-psbA gene region 14 The ITS4-5, matK, trnW-psaJ, trnSGCU-trnGGCC gene regions and combinations of ITS4-5 + matK, ITS4-5 + trnW-psaJ, ITS4-5 + trnSGCUtrnGGCC, rbcL + matK + Rpl32-trnL + trnW-psaJ + trnSGCU-trnGGCC obtained positive values for the statistical analyzes D, Fs, and D*, F* showed that lotus samples occurred both low frequency and The high frequency of the number of polymorphic sites compared with the expectation is neutral, the lack of rare alleles on the nucleotide sequences of the gene regions obtained from 33 lotus samples (Table 3.3) In recent times, the researched lotus samples have undergone a selection process in the direction of equilibrium, leading to a decrease in the number of lotus samples at the time of research Table 3.3 The results of testing the neutrality Gene region ITS4-5 rbcL matK trnH-psbA Rpl32-trnL trnW-psaJ trnSGCU-trnGGCC ITS4-5 + rbcL ITS4-5 + matK ITS4-5 + Rpl32-trnL ITS4-5 + trnW-psaJ ITS4-5 + trnSGCU-trnGGCC rbcL + matK + Rpl32-trnL + trnW-psaJ + trnSGCU-trnGGCC D 2,291* -1,1404* 0,3664* -0,7924* -2,1793* 1,6264* 1,5804* 1,6484* 0,5434* -0,2614* 2,498* 2,484* D* 1,1354* -1,7134* 1,916** 0,5844* -3,7212* 0,5844* 0,5844* 0,4154* 1,9242* -1,9074* 1,207 4* 1,2074* F* 1,724* -1,7904* 1,648* 0,2314* -3,7972* 1,0074* 0,9924* 0,9194* 1,724* -1,6294* 1,8632* 1,8582* Fs 7,078 -1,290 21,765 -0,521 1,375 1,743 1,712 4,867 20,616 5,628 4,049 4,036 S 0,009 0,977 0,927 0,549 0,458 0,465 0,037 0,020 0,061 0,062 0,1544* 1,2244* 1,0204* 13,157 Note: Statistical significance: *, p < 0.05; Statistically significant: 2*; p < 0.02; Statistically significant: 3*; p < 0.01; not statistically significant: 4*, p > 0.10; D, Tajima's statistic; Fs, Fu's statis-tic; S, Strobeck's statistic; D * and F *, Fu and Li's statistics Meanwhile, the gene regions rbcL, Rpl32-trnL, trnH-psbA, and gene region combination ITS4-5 + Rpl32-trnL showed the number of polymorphic nucleotide positions compared with the expectation at the neutral level occurring with frequency low number among studied lotus samples The superior number of rare alleles has made the lotus individuals in the two 15 groups of lotuses tend to split due to the change after the bottleneck event under the influence of the selective scanning process or genetic hitchhiking causing the number of lotus populations to tend to increase at the time of the study These statements are expressed through negative and positive values obtained from statistical analyzes D, Fs, and D *, F * (Table 3.3) The results of probabilistic analysis of observing the entire range of haplotypes in any random selection show that most of the gene regions and the combinations of marker gene regions obtained from 33 lotus samples have low "S" values (S < 0.5), ranging from 0.000 to 0.465 The results show that some new haplotypes are dominant in the number of studied lotus samples and the probability of getting all these haplotypes is low by random selection The gene regions rbcL, trnH-psbA, Rpl32-trnL obtained high "S" values, ranging from 0.549 to 0.977, indicating the appearance of some rare haplotypes containing only or lotus samples with large genetic differences compared with the remaining lotus samples (Table 3.3) 3.3 The genetic characteristics analysis between the two group of white and pink lotus based on a combination of chloroplast marker gene regions The % (G + C) contained in the combination of chloroplast gene regions did not have a big difference between the two groups of lotus, 33.4% (white lotus) and 33.5% (pink lotus) The results of the analysis of genetic parameters k, π, S, Eta in the white lotus group showed that these values were lower than in the pink lotus group The white lotus group was divided into different haplotypes, with Hd = 0.618, and the pink lotus group was divided into different haplotypes, with Hd = 0.818 (Table 3.4) Bảng 3.4 The results of genetic characteristics analysis between the two group of white and pink lotus n % (G+C) S Eta h Hd k π (x10-3) Group 33,400 5 0,618 1,564 0,180 White lotus 11 33,500 84 85 0,818 29,082 3,280 Pink lotus 22 The results of D, D*, and F* statistical analysis for the white lotus group showed that the number of polymorphic nucleotide positions compared with the expectation at neutral occurred with low frequency among lotus samples 16 Recently, the combined nucleotide sequence of the gene regions has appeared that many rare nucleotide variants have increased the number of haplotypes, and the number of populations tends to increase due to the subdivision small of the number of lotus samples in the group lotus studied under the impact of background selection These statements are expressed through negative values obtained from statistical analyzes D, D *, and F * and positive values obtained from Fs (Table 3.5) Meanwhile, in the pink lotus group, the lotus samples had both low-frequency and high-frequency phenomena in the number of polymorphic sites compared to the expected neutral level, there was a shortage of rare alleles on the sequence of a combination of chloroplast marker gene regions in 33 lotus samples In recent times, the lotus samples have undergone a rigorous selection process, leading to a decrease in the number of lotus samples at the time of research These statements are expressed through positive values obtained from D, D *, and F * statistics (Table 3.5) Table 3.5 The test results for neutrality between the two group of white and pink lotus Fs D D* F* S Group 1,545 - 0,322* - 0,764* - 0,736* 0,446 White lotus 14,465 0,993* 1,134* 1,277* Pink lotus Note: *: Not significant, p > 0,10 The probability of observing the entire range of haplotypes under any random selection of the two lotus groups obtained a low S value (S < 0.5), ranging from to 0.446 (Table 3.5) The Fst value with the largest genetic distance observed between the two lotus groups was 0.00233 (p < 0.05) (Table 3.6) This value shows that the genetic difference is not significant between the two studied groups of lotus collected at different locations in Hue Table 3.6 The result of Fst value between the two group of white and pink lotus White lotus Pink lotus Group 0,00032 White lotus 0,00233* Pink lotus Note: Fst value is significant at p < 0.05; -: No data 17 3.4 The analysis of the evolutionary process of some lotus samples grown in Hue based on 10 marker gene regions 3.4.1 Nuclear gene region marker The results of the phylogenetic tree built based on the ITS4-5 marker gene region showed that the lotus samples belonging to the same group were genetically related to each other and separated into a separate evolutionary branch (Figure 3.6) Figure 3.6 Phylogenetic tree of lotus varieties samples collected based on the ITS4-5 gene region some lotus samples grown in Hue 3.4.2 Chloroplast gene region marker The results of building a phylogenetic tree based on individual chloroplast marker gene regions showed that 33 lotus samples all had genetic differentiation that was not clearly shown when they divided into different branches, except Concave white lotus ST02 and High-yield-pink SH09 (Appendix 5.1 to 5.8) Figure 3.7 Phylogenetic tree based on a combination of marker gene regions nucleotide differences between some lotus samples grown in Hue The researched lotus samples have a more obvious division of evolutionary branches, appearing many haplotypes containing only one lotus sample in the studied lotus group when there is a combined analysis of one or more nucleotide 18 sequences of the marker gene regions together such as ITS4-5 + trnW-psaJ, ITS4-5 + trnSGCU-trnGGCC, ITS4-5 + rbcL, ITS4-5 + Rpl32-trnL, rbcL + matK + Rpl32-trnL + trnW -psaJ + trnSGCU-trnGGCC and the combination rbcL + matK + trnH-psbA + accD-psaI + ndhA + psbE-petL + Rpl32-trnL + trnW-psaJ + trnSGCU-trnGGCC (Appendix 5.1 to 5.8, Figure 3.7 and Figure 3.8) Figure 3.8 Phylogenetic tree based on a combination of chloroplast marker gene regions among some lotus samples grown in Hue The samples High-yield-pink SH06 and SH09 are distributed into separate sub-branches and have different and larger genetic distances than pink lotus samples distributed in the same clade, in which samples Highyield-pink SH09 have the greatest genetic distance The sample Concave white lotus ST02 split into a subgroup and had a larger genetic distance than the remaining white lotus samples distributed in the same clade The sample of Concave white lotus ST02 belonging to the local lotus group was collected at Tinh Tam lake, Thuan Loc ward, Hue city For people who grow lotus for economic benefits, white lotus is the preferred lotus variety because it can both exploit flowers and seeds at a very high price White lotus seeds are famous for their delicious taste that other lotus varieties not have On the other hand, According to Nguyen Thi Quynh Trang et al (2017), authors also determined that the White Concave white lotus variety is a local lotus variety with value in flower color, yield, quality, and potential production produced for conservation, exploitation, and development Besides, according to the research of Le Cong Son (2008), the White Lotus variety is a typical variety 19 of Hue, with white flower color, long flower buds, pointed tip, flat mirror, or concave, small but many seeds It is also focused on planting in Hue relics in the inner city, of Hue city Because of those superior characteristics, and the specific difference in genetic distance based on the sample of Concave white lotus ST02 based on the nucleotide sequences of 10 marker gene regions compared to other lotus samples in the study, I selected a sample of the Concave white lotus ST02 to harvest lotus seeds as raw materials for the extraction, isolation, and evaluation of biological activities of pure compounds is very necessary to serve the potential exploitation the capacity of lotus plants and the conservation of precious lotus varieties 3.5 Survey results of total solvent extracted from the seeds of the Concave white lotus variety In 100 g of raw materials of Concave white lotus seed powder raw materials, the Content of total crude extraction was extracted in water and ethanol solvents with varying concentrations were different, ranging from 0.604 to 1.703 g In, ethanol solvent at 70% concentration gave the collection of the total crude extraction and a group of alkaloid and flavonoid compounds the highest, accounting for 1.703 g (Table 3.7) Table 3.7 The results of total crude extraction and qualitative alkaloids and flavonoids from the seeds of the Concave white lotus Solvent Content of total crude extraction (g) Alkaloid Flavonoid Water 0,865c ± 0,057 + + Ethanol 30% 1,142d ± 0,061 + + Ethanol 50% 1,161d ± 0,009 ++ ++ Ethanol 70% 1,703e ± 0,068 +++ +++ Ethanol 90% 0,783b ± 0,009 ++ ++ Ethanol 96% 0,604a ± 0,011 ++ ++ Note: +: weak expression; ++: average expression; +++: strong expression; Different letters in the same row indicate a statistically significant difference with p < 0.05 3.6 The results quantitative of alkaloids and flavonoids in extracts of Concave white lotus seeds The quantitative results of alkaloids and flavonoids shown in Table 3.8 showed that the n-Bu fraction gave the highest alkaloid content, accounting for 20 209.667 mg of highly purified NU/g and the NE fraction gave the highest flavonoid content, accounting for 414,300 mg CE/g pure high purity (Table 3.8) Bảng 3.8 The results of quantification of alkaloids and flavonoids in crude and fractionated extracts of Concave white lotus seeds Fractionated extracts Total flavonoids (mg CE/g) Total alkaloid (mg NU/g) Cao tổng số 19,790b ± 0,385 0,718a ± 0,041 d NE 414,300 ± 1,500 52,571d ± 0,025 c n-Bu 118,000 ± 11,456 209,667e ± 0,041 a NP 9,050 ± 0,250 6,676b ± 0,004 a P 6,550 ± 0,250 6,771c ± 0,019 3.7 Results of the isolation of compounds from Concave white lotus seeds Results isolate in PD3, PD4, and PD5 obtained compounds with symbols NH2 (136 mg), NH8 (150 mg), and NH11 (217 mg) The obtained Rf values are 0.651, 0.724, and 0.651, respectively All three compounds gave the characteristic red-brown color of alkaloids when dyed with Bouchardat and dried at 105oC (Table 3.9) Table 3.9 Results of properties of compounds isolated from n-Bu fractions of Concave white lotus seeds Compound Weight symbol (mg) NH2 136 NH8 150 NH11 217 Solvent system Reagents CHCl3:Acetone:MeOH Bouchardat (50:50:0,5) CHCl3:MeOH:NH4OH Bouchardat (96:4:0,5) CHCl3:MeOH:NH4OH Bouchardat (96:4:0,5) Rf Color expression Red 0,651 brown Red 0,724 brown Red 0,651 brown The results of LC-MS/MS data analysis including retention time, molecular ions, and some important fragmented ions are presented in Table 4.10 as follows: High-resolution mass spectrometry recorded positive ion [M+H]+ of compounds NH2, NH8, and NH11 with m/z = 318,9372, m/z = 314.1750 and m/z = 266.1595, respectively [C 19H21NO2 + H]+, [C19H23NO + H]+ and [C17H15NO2 + H]+ consistent with a molecular formula NH2:C19H21NO2, NH8:C19H23NO3 and NH11: C17H15NO2 (Table 4.10) The results of MS/MS analysis of ion fragmentation in positive ion measurement mode are listed in table 3.10 When comparing and contrasting 21 the NIST2017 and reference by Deng et al (2016), Xu et al (2011), Zou et al (2019), Tan et al (2021), Lima et al (2020), Gontijo et al (2018) analysis compounds this, We can determine the compounds- NH2 is nuciferine, NH8 is armepavine and NH11 is anonaine (Table 3.10) Table 3.10 The results of mass spectrometry analysis of the pure compounds Compo Retenti Molecula und on time r symbol (TR, Formula min) Predicted [M+H]+ [M+H]+ Com- Molecula Error pound r Weigh (ppm) Name NH2 12,065 C19H21NO2 296,971 >5 NH8 5,686 C19H23NO3 Armepavine 314,175 1,9 NH11 9,620 >5 C17H15NO2 Nuciferine Anonaine 266,160 m/z MS/MS 233,9; 250,1; 265,2; 296,2 107; 145,2; 173,9; 189,3; 239,1; 251,1; 268,2; 314,1 178,3; 189; 190; 191; 219 3.8 Biological activity test results 3.8.1 Antioxidant activity The results showed that at the concentration of 100 mg/mL, the DPPH free radical scavenging ability of the 70% total ethanol extract was the highest at 82,333 %, corresponding to the IC50 = 6.931 mg/mL (Table 3.11) Table 3.11 The DPPH free radical scavenging activity of 70% total ethanol extract from the Concave white lotus seeds Reagent concentration (mg/mL) 100 50 25 12,5 IC50 Concave white lotus 82,333d ± 0,577 65,000c ± 2,000 56,333b ± 1,527 51,667a ± 1,155 6,931 ± 2,614 Standard concentration (µg/mL) 10 0,4 0,08 IC50 Acid ascorbic 99,333d ± 1,155 67,667c ± 1,528 51,333b ± 2,527 15,667a ± 3,512 1,734 ± 0,151 At a concentration of 100 µg/mL, nuciferin was the highest DPPH free radical scavenging activity substance (mean = 94.620%), followed by armepavine (74.019%) anonaine was the lowest free radical scavenger activity substance (61.151%) The IC50 values obtained from these 22 substances were 30.031 µg/mL (nuciferin), 43.093 µg/mL (armepavine), and 68.217 µg/mL (anonaine), respectively (Table 3.12) Table 3.12 The DPPH free radical scavenging activity of pure compounds Concentr ation (µg/mL) 100 20 0,8 IC50 Compounds Nuciferine Armepavine Anonaine Acid ascorbic 94,620d ± 0,657 46,894c ± 3,329 35,587b ± 2,725 25,916a ± 1,340 30,031 ± 1,506 74,019c ± 2,400 39,827b ± 3,324 33,750b ± 6,236 16,722a ± 3,133 43,093 ± 14,215 61,151d ± 2,349 37,368c ± 2,028 27,928b ± 3,975 15,003a ± 2,256 68,217 ± 6,214 99,483d ± 0,896 68,172c ± 1,561 51,822b ± 2,607 16,059a ± 3,414 1,734 ± 0,151 Note: Ascorbic acid is active over a concentration range of 0.08; 0.4; and 10 µg/mL 3.8.2 Cytotoxic activity assay against some cancer cell lines For the 70% total ethanol extract, the cytotoxicity in vitro was level mean with IC50 = 37.330 µg/mL to 61.06 µg/mL on 10 cell lines examined, in which the best inhibitory effect on human prostate cancer cell line LNCaP with IC50 = 37.330 µg/mL (Table 3.13) Table 3.13 IC50 values of 70% total ethanol extract Cell line LNCaP HepG2 MCF7 Hela SK-LU-1 MKN7 SW480 KB SK-Mel2 HL-60 HEK-293A Total ethanol 70% extract IC50 (µg/mL) 37,330 ± 3,950 54,110 ± 5,580 47,550 ± 0,410 48,020 ± 3,660 52,410 ± 5,370 57,450 ± 5,020 50,490 ± 3,740 61,060 ± 6,530 56,450 ± 2,310 45,990 ± 4,280 >100 Standard Ellipticine IC50 (µg/mL) 0,450 ± 0,010 0,490 ± 0,020 0,480 ± 0,040 0,410 ± 0,020 0,360 ± 0,020 0,400 ± 0,040 0,440 ± 0,050 0,450 ± 0,030 0,480 ± 0,050 0,410 ± 0,040 0,40 ± 0,020 The cytotoxicity results of three active compounds isolated from the Concave white lotus seeds showed the did no activity of the investigated substances at the studied concentrations The IC 50 value cannot be determined in the concentration range from 0.8; 4; 20 and 100 µg/mL, Ellipticine positive control was stable in the experiment (Table 3.14) 23 Table 3.14 IC50 values of pure compounds Compounds (µg/mL) MKN7 Nuciferine >100 Armepavine >100 Anonaine >100 Ellipticine 0,360 ± 0,030 Cell line KB >100 >100 >100 0,360 ± 0,030 SK-Mel-2 >100 >100 >100 0,300 ± 0,030 HEK-293A >100 >100 >100 0,40 ± 0,020 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 4.1 Conclusions From the research results of the thesis, I have drawn the following conclusions: - The determined size of the marker gene regions obtained from 33 different lotus samples in Hue ranged from 350 to 1667 bp The two gene regions ITS4-5 and trnH-psbA varied in size between the studied lotus samples, ranging from 729 to 744 bp (ITS4-5) and from 351 to 410 bp (trnH-psbA) Lotus samples have a high degree of similarity with N nucifera species - The nucleotide sequences of the marker gene regions were obtained from 33 lotus samples deposited on GenBank with codes ITS4-5 (MT903421 to MT903453), rbcL (MN011708 to MN068956), matK (MN011719 to MN068978), trnH- psbA (MN011730 to MN086252), accD-psaI (MN086253 to MN086285), psbE-petL (MT901764 to MT901796), Rpl32-trnL (MT901731 to MT901763), trnW-psaJ (MT905225 to MT905257), trnSGCU-trnGGCC (MT905258 to MT905290) and ndhA (MZ611976 to MZ612008) - The determined gene markers out of 10 gene regions used in the study had nucleotide differences between 33 lotus samples grown at different locations in Hue The gene regions showing the difference according to a general rule among lotus samples expressed through the respective nucleotide positions are ITS4-5: 404 (C-T), 625 (G-A) and 626 (A-G), 408 (G-T) ) and 576 (C-A); rbcL: 459 (G-T); trnW-psaJ: 504 (T-G); trnSGCU-trnGGCC: 369 (A0) and 545 (A-T); Rpl32-trnL: 885 (C-G), 895 (CATC-ATCA), 920 (A-G) and 927 (A-G); matK: 293 (G-A), 323 (G-A-T), 709 (G-T) and 819 (T-G) and the number of ATAAA repeats of the trnH-psbA gene region 24 - Determining the phylogenetic tree based on the combination of marker gene regions of the chloroplast genome with nucleotide sequence difference between 33 lotus samples grown in Hue From there, a sample of the Concave white lotus ST02 was selected, which is an indigenous lotus variety of Hue, has a distinctive genetic relationship, has a larger genetic distance than the remaining lotus samples, and is isolated into a separate evolutionary branch This lotus sample was selected as a raw material source in the study of extracting pure compounds and evaluating the biological activities of compounds extracted from lotus seeds - The 70% total ethanol extract produced from the seeds of the Concave white lotus variety demonstrated excellent antioxidant activity, with an IC 50 = 6.931 mg/mL for DPPH free radical scavenging ability On 10 cancer cell lines, the cytotoxic activity at 50% (IC 50) was between 37.330 µg/mL to 61.06 µg/mL on average The human prostate cancer cell line LNCaP had the best cytotoxic action, with an IC 50 = 37.330 µg/mL - Three pure compounds were isolated from the n-Bu fraction from Concave white lotus seeds with an alkaloid nature: nuciferine (NH2:C19H21NO2), armepavine (NH8:C19H23O3N), and anonaine (NH11:C17H15NO2) The DPPH free radical scavenging activity was high on all compounds, respectively, the IC50 obtained for each compound was 30.031 µg/mL (nuciferine), 43.093 µg/mL (armepavine) and 68.217 µg/mL (anonaine) The toxic activity of pure compounds when tested on cancer cell lines MKN7, SK-Mel-2 and KB did not show, and the inhibitory value at 50% (IC50) could not be determined in the concentration range degrees from 0.8; 4; 20 and 100 µg/mL 4.2 Reconmendations From some of the results obtained, I make the following recommendations: - Increasing the number of gene regions, and increasing the number of samples of lotus to be able to more objectively and accurately assess the genetic difference of the lotus grown in Hue- Continue to isolate and test biological activities for compounds present in Concave white lotus seeds 25 [1] [2] [3] [4] [5] [6] DANH MỤC CÁC CƠNG TRÌNH ĐÃ CƠNG BỐ Dang Thanh Long, Hoang Thi Kim Hong, Nguyen Thị Quynh Trang, Le Ly Thuy Tram, Nguyen Van Hiep, Nguyen Phan Thuy Tien (2019), “Genetic diversity analysis of Lotus species (Nelumbo nucifera gaertn.) in Thua Thien Hue based on ITS4-5 genetic region”, Plant Cell Biotechnology and Molecular Biology, 20(23&24), pp 1160-1171 Dang Thanh Long, Hoang Thi Kim Hong, Nguyen Thị Quynh Trang, Le Ly Thuy Tram, Nguyen Phan Thuy Tien; Nguyen Thi Ngoc Hanh (2020), “Study of the procedure for bath ultrasound-assisted extraction of total flavonoid from Lotus seeds and testing some biological activities”, Indian Journal of Agricultural Research, 54(5), pp 563-570 Dang Thanh Long, Hoang Thi Kim Hong, Le Ly Thuy Tram, Nguyen Thi Quynh Trang (2021), “Evalution of genetic diversity by DNA barcoding of local Lotus populations from Thua Thien Hue province”, Indian Journal of Agricultural Research, 55(2), pp 121-128 Dang Thanh Long, Hoang Thi Kim Hong, Le Ly Thuy Tram (2022), “ Research on phylogenetic relationship of Lotus populations collected in Thua Thien Hue province, Vietnam based on the chloroplast genome by DNA barcode,” Indian Journal of Agricultural Research, 56(3), pp 249-254 Dang Thanh Long, Hoang Thi Kim Hong, Le Ly Thuy Tram (2022), “Analysis genetic variation and phylogenetic relationships of local Lotus collecred in Thua Thien Hue province, Vietnam by DNA barcoding”, Resaarch Journal of Biotechnology, 17(2), pp 48-56 Long Dang Thanh, Quang Hoang Tan, Trang Nguyen Thi Quynh and Hoang Thi Ngoc Han (2022), “Study on the isolation and bioactivity assay of some compounds from Nelumbo nucifera Gaertn seeds collected at Tinh Tam Lake, Thua Thien Hue, Vietnam”, Plant Cell Biotechnology and Molecular Biology, 23(21&22), pp 42-53