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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

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 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

2 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, anti-amnesia, 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

2 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

3 Noval contributions

- The project has contributed new results as follows:

1 Constructed a data set of nucleotide sequence differences of 1 marker gene region from the nuclear genome and 9 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

2 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

3 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)

4 Initially, these three compounds had strong antioxidant activity but did not show cytotoxic activity against three cancer cell lines MKN7, SK-Mel-2 and KB at all the treatment concentrations

4

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 1 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 2 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 1 to 2 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)

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)

6 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-2-picrylhydrazyl (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 FBS Cells were transplanted after 3-5 days at a ratio (1:3) and kept in incubators at 37C, 5% CO2

serum-c Determination of cancer cytotoxicity against monolayer cultured cells: The assay was carried out to determine the total protein content based on the

7 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 60): Method using tetrazolium salts as a reagent in the colorimetric assay, thereby assessing cell survival and viability

(HL-2.2.3 Data analysis methods: Experimental experiments were repeated 3

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 3 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)

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 trnS GCU -trnG GCC

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 MT903453), 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), trnS GCU -trnG GCC (MT905258 to

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%

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

region

Size (bp)

Number of monomorphic sites

Note: S: Number of variable sites; Eta: Total number of mutations

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 0 to 75 positions, corresponding to the percentage of polymorphic nucleotides that appeared on nucleotide sequences in gene regions ranging from 0 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), 0 to 75 (S) and 0 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 = 1 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), trnS GCU

-trnG GCC (k = 0.504 and π = 0.460 x10-³), psaJ (k = 0.511 and π = 0,700 x10-³) and matK gene region, these values were

trnW-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 1 to 5 haplotypes, corresponding to the obtained

Hd ranging from 0 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 trnS GCU trnG GCC contain 1 to 2 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

Hue A: gene regions rbcL, B: gene regions Rpl32-trnL, C: gene regions

trnW-psaJ và D: gene regions trnS GCU -trnG GCC

A B C D

12 The sample high-yield lotus SH09 has 7 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 7 mutant sites was formed

with the distribution of lotus samples in haplotypes being different, appearing that one haplotype contained only one sample of sample high-yield lotus SH09 (Table 3.1) The results of nucleotide differences in the

sequences of each gene region rbcL, trnW-psaJ, and trnS GCU -trnG GCC

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

7 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 2 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