Application of molecular marker start codon target (scot) in individual identification of cat hoa loc mango (mangifera indica l)

NGUYEN TAT THANH
UNIVERSITY
/TVgaSV*

NGUYEN TAT THANH

True learning - True practice - True success - True future

FACULTY OF BIOTECHNOLOGY

GRADUATION THESIS

APPLICATION OF MOLECULAR MARKER:
START CODON TARGET (SCOT) IN
INDIVIDUAL IDENTIFICATION OF CAT HOA
LOC MANGO (Mangifera indica L)

Student’s name

: Pham Thi Hoa Mai

Student ID

:1611540685

Supervisor

: MSc. Nguyen Thi Nha

Ho Chi Minh City, 2020

TABLE OF CONTENTS

ACKNOWLEDGEMENTS.................................................................................................. i

TABLE OF CONTENTS..................................................................................................... ii

ABSTRACT...........................................................................................................................iv
LIST OF FIGURES............................................................................................................... V

LIST OF TABLES................................................................................................................ vi
LIST OF ACRONYMS...................................................................................................... vii

INTRODUCTION............................................................................................................. viii

CHAPTER 1. LITERATURE REVIEW........................................................................ 1
1.1 Introduction of Cat Hoa Loc Mango tree..................................................................... 1

1.1.1 Ecological characteristics............................................................................................ 1

1.1.2 Distribution place.........................................................................................................2
1.1.3 The values of Cat Hoa Loc mango.............................................................................2

1.2 Techniques used in research.......................................................................................... 3
1.2.1 Molecular marker technique........................................................................................3
1.2.2 Molecular marker SCoT..............................................................................................3
1.3 Research situation of SCoT molecular indicator and application of molecular

indicator in individual identification..................................................................................... 4
1.3.1 Studies ofSCoT directive application....................................................................... 4

1.3.2 Application of molecular markers in individual identification.............................. 7
CHAPTER 2. CONTENTS AND METHODS............................................................. 9

2.1 Place of administration....................................................................................................9
2.2 Contents............................................................................................................................ 9
2.3 Methods............................................................................................................................ 9

ii


2.3.1 DNA extraction............................................................................................................ 9
2.3.2 Screening polymorphic SCoT markers................................................................... 12

2.3.3 PCR data analysis....................................................................................................... 14

CHAPTER 3. RESULTS AND DISCUSSION............................................................17

3.1 Sample collection.......................................................................................................... 17
3.2 DNA extraction.............................................................................................................. 20
3.3 Evaluate the polymorphism of 27 primers on 15 individuals of Hoa Loc mango ..21

3.4 Identify specific DNA bands to demonstrate certain individuals...........................27

CONCLUSIONS AND RECOMMENDATION .......................................................... 29
REFERENCES.................................................................................................................... 30
APPENDICES...................................................................................................................... 32

iii

ABSTRACT
The study “Application of molecular marker: start Codon Target (SCoT) in

individual identification of Cat Hoa Loc mango (Mangiferaindica £)" was carried out
from October 2019 to July 2020 in Molecular Biology lab, Faculty of Biotechnology,
Nguyen Tat Thanh Univerisity with an aim to based on Start Codon Target (SCoT)

marker system.
A total of 15 Cat Hoa Loc mango cultivars included in the study were performed

DNA extraction, polymorphic selection, and amplification of SCoT markers, and
genetic diversity analysis using NTSYSpc software. The polymorphism of 27 primers
on 15 individuals of Hoa Loc mango was evaluated. The study resulted in the successful
identification of specific DNA bands for 15 individuals.

IV


LIST OF FIGURES
Figure 1.1 Picture of Cat Hoa Loc mango............................................................................ 1
Figure 2.1 Preparation and data entry in Microsoft Excel.............................................. 15
Figure 2.2 Example of similarity matrice of sample...................................................... 15
Figure 2.3 Example of dendrograms drawing (phylogenetic trees)..............................16
Figure 3.1 Total DNA on electrophoresis gel................................................................. 20
Figure 3.2 PCR products of SC0TO6, SCoT14, SC0TI8, and SC0T44 across 15 Cat
Hoa Loc mango cultivars......................................................................................................23

Figure 3.3 Similarity matrix of 27 polymorphic SCoT marker across 15 Hoa Loe
cultivars using SM coefficient............................................................................................. 24

Figure 3.4 Phylogenetic tree of 27 polymorphic SCoT marker across 15 Cat Hoa Loc
mango cultivars...................................................................................................................... 24

Figure 3.5 Similarity matrix of 27 polymorphic SCoT marker across 15 Hoa Loc
cultivars from SOFR1 using SM coefficient...................................................................... 26

Figure 3.6 Phylogenetic tree of 27 polymorphic SCoT marker across 15 Cat Hoa Loc
mango cultivars from SOFRI.............................................................................................. 26

Figure 3.7 PCR products of SCoT19 across 15 Cat Hoa Loc mango cultivars from
SOFRI..................................................................................................................................... 27

V


LIST OF TABLES
Table 2.1 Sequences of SCoT markers using in this study............................................. 12
Table 2.2 Component content of PCR reaction............................................................... 13
Table 2.3 PCR cycling conditions..................................................................................... 13
Table 3.1 List of mango cultivars using in this study....................................................17
Table 3.2 OD concentration of 15 DNA samples.......................................................... 20
Table 3.3 List of polymorphic SCoT markers selected..................................................22
Table 3.4 List of SCoT identified cultivars..................................................................... 27

vi


LIST OF ACRONYMS
bp

Base pair

DNA

Deoxyribonucleic acid

SCoT

Start Codon Target

UPGMA

Unweighted Pair Group Method with Arithmetic mean

Rnase

Ribonuclease

PLS

Plant Lysis buffer

PBB

Plant Binding buffer

PWB

Plant Wash buffer

EB

Elution Buffer

PCR

Polymerase Chain Reaction

uv

Ultraviolet

TBE

Tris-Borate - EDTA

OD

Optical Density

Vil


INTRODUCTION
1. Rationale for this thesis
Identifying varieties is very important in choosing which varieties of plants to take

the best advantages of the natural conditions and other resources of the region. The best

way to utilise that advantages is choosing the plants with the most favorable conditions
for them to nuture, develop, and produce high yields. Besides, identification of the

varieties is important to the management and exploitation of its use-value. Identifying
varieties with agronomic properties such as appearance observing, color, taste, ... still
faces with many difficulties and inaccuracy when identifying seed samples with similar

characteristics in shapes and sizes.
Molecular technology has appeared and become an effective application for the
selection of plant varieties, genomic, genetic diversity, and phylogenetic research in
many types of plants. Several crops such as tomato, rice, maize, wheat, soybean that are

resistant to pests, salinity, drought with high productivity have been successfully
selected with the help of molecular markers.
Currently, compared to other methods to evaluate genetic diversity, molecular
techniques help US to do the task easier and faster. More and more researches apply

molecular markers in the study of plant molecular genetics. In particular, the SCoT
molecular marker technique is a simple technique based on the PCR reaction of
amplifying DNA fragments that contain the ATG codon. SCoT advantages includes

highly polymorphic amplification which is related to functional genes, ability to perform
reactions without genetic information, and application in genetic diversity analysis, QTL

mapping as well as bulked segregant analysis.
The study of using SCoT on mangoes in Vietnam has been conducted, besides the

genetic grouping for 30 cultivar samples, this study has discovered some specific DNA
bands that can be used to identify individuals of mango.

Among the current mango varieties grown in Vietnam, Cat Hoa Loc mango

(Mangiferci indica L) is a fruit food favored by both its taste and the nutritional value.
This mango is high in nutrients such as calcium, potassium, carbohydrates,
phosphorus, magnesium, vitamins such as vitamins A, and c. Besides, Hoa Loc

viii


mango has a high economic value in exporting big markets such as the United States,
Singapore, China, Europe, Korea, Japan, Australia, New Zealand, etc. The

identification of Cat Hoa Loc mangoes is very crucial in the management and
exploitation of the use-value of Hoa Loc mangoes.

2. Objectives
The aim of this study is to identify specific DNA bands that identify certain

individuals and evaluate the polymorphism of 27 primers on 15 individuals of Hoa Loe

mango.

IX


Chapter 1. Literature review

CHAPTER 1. LITERATURE REVIEW
1.1 Introduction of Cat Hoa Loc Mango tree
Scientific name and location of Mango in the classification system:

Kingdom

Plantae

Phylum

Mangoliophyta

Class

Mangoliopsida

Order

Sapindales

Family

Anacardiaceae

Genus

Mangifera

Species

Itĩdica L

Figure 1 Picture of Cat Hoa
Loc mango

Hoa Loc mango was first grown in Hoa Loc village, Giao Duc district, Dinh Tuong

province in 1930 (now Hoa hamlet, Hoa Hung village, Cai Be district, Tien Giang
province).

Mango characteristics involve moderate growing plants, oblique branches,
umbrella-shaped canopy, oblong leaves, wavy leaf covers, pointed tail, 400 - 600 grams
in weight of fruit, oblong fruit, near-inflated stems, bright yellow skin when ripping, 28

- 32 mm in thick and fleshy fruit, 78 - 80 % edible fleshy rate, low fiber, firm and

smooth flesh, a sweet bar, Brix degree from 20 - 22 %, 105 - 120 days in flower-tofruit process from, good drought tolerance L

1.1.1 Ecological characteristics
Cat Hoa Loc Mango has average growth and development rate. Suitable soil is

alluvial soil in the riverside, rich in nutrients. Besides, Cat Hoa Loc mango can also be
grown on acidic or saline soils.
The appropriate temperature for growing Hoa Loc mangoes is between 18 - 35 °C

but still tolerant at temperatures of 0 °C and above 40 °C for several weeks. Terrain
altitude 1300 m, where precipitation is about 1000 mm or more, however, 2-4 month
drought period will help in fruiting better.

1


Chapter 1. Literature review

Prevent cardiovascular disease: Mango is an excellent fruit that provides vitamins

-

A, E, and selenium to the body and helps fight heart disease.
-

Improve memory: Mangoes contain glutamine acid, a substance known to

improve memory and keep brain cells active.
Enhance eyesight: The vitamin A component of mango will be an important

-

nutrient to develop the vision, prevent night blindness and dry eye.

-

Enhancing fertility,...

1.2 Techniques used in research

1.2.1 Molecular marker technique
Molecular markers are the kind of markers that indicate the gulfs between different
species based on the differences in their DNA, protein, and enzyme molecules.

Molecular markers are divided into several categories grounded on the differences in
methods and techniques in determining polymorphism.

DNA markers are the most widely used due to great varieties of them. DNA

markers are made up of different types of DNA mutations such as substitutions (point
mutations), rearrangements (addition or subtraction of nucleotides), or errors in

replicating adjacent DNA segments. DNA markers are not often located in transcription
areas.

DNA markers are widely used in kinds of studies involving genetic mapping,

breeding selection, evaluation of genetic diversity, cultivar identification, selection for
disease resistance traits, resistance to adverse environmental conditions, genetic studies

of yield and quality, and phylogenetics3.

1.2.2 Molecular marker SCoT
The Start Codon Targeted markers - SCoT is a DNA marker technique for

polymorphism detection grounded on PCR reaction with 15 to 18 nucleotide primers,
which were first published by Collard and Mackill in 2009, were designed based on high

conservation sequences around the ATG opening codon of plants4.

3


Chapter 1. Literature review

The SCoT marker has similarities with the Random Amplification of Polymorphic

DNA (RAPD) techniques and Inter Simple Sequence Repeat (ISSR) techniques because

all of them use only one DNA fragment for both forward and reverse primers 5.
The difference of SCoT technology from other techniques is that it uses the highly

conserved fragments around the ATG codon in the plant genome to perform PCR, which

makes the SCoT markers get high sensitivity for amplifying plant genes as well as being

able to relate to functional and specific plant genes. At the same time, SCoT markers
can be used to determine polymorphism at an individual level without genome
information of the species. Low - cost and easy - performed advantages are the points

that make SCoT more elite than other molecular markers.
The SCoT marker is published to be highly effective in applying quantitative trait

mapping (QTL map), bulked segregant analysis, especially in genetic diversity research.

SCoT can also be combined with RAPD and SSR markers at the same time in genetic
analysis in plants6.
The SCoT markers have been successfully implemented in analyzing genetic

diversity in many subjects including mango 4, sugar cane 7, wheat8, medicinal plants9,
and others.

1.3 Research situation of SCoT molecular indicator and application of molecular

indicator in individual identification

1.3.1 Studies of SCoT directive application
In 2009, SCoT was first designed and published by Collard and Mackill after they

realized the disadvantages of the RAPD markers such as genomic information

requirements and low - reproducible ability. The SCoT markers appeared to solve these

difficulties and were successfully tested on rice '°.
In 2013, Jian-Ming Wu at al. analyzed genetic diversity in sugarcane in China
based on the SCoT markers system. In this study, 20 SCoT primers were designed to

evaluate genetic diversity among 107 sugarcane varieties. These primers amplified 176

DNA band patterns, of which 163 polymorphic bands (92.85 %). The value of genetic

polymorphism (PIC) ranges from 0.783 to 0.907 with an average value of 0.84. The

ƯPGMA method divided 107 sugarcane varieties into 6 groups with an average genetic
4


Chapter 1. Literature review

similarity coefficient at 0.674. High genetic diversity was found on three main sugarcane
cultivars including ROC22, ROC 16, and ROCIO (accounting for about 80 % of the total

sugarcane area in China). Results of genetic diversity among local sugarcane varieties

provides basic data for the management of sugarcane cultivars, as well as, development

of genetic collections, regional distribution, and grading 7.
In 2014, the SCoT marker system was used in determining fruit quality

characteristics and genetic comparative analysis between 20 mango varieties (15 local
varieties and 5 common varieties) in India. With 80 SCoT markers used, they

successfully selected 19 makers to amplify. These primers produced 117 bands on 20
samples with 96 polymorphic bands (79.57 %) with an average of 5.05 polymorphism

band per primer. 17 of 19 used primers created 34 cultivars - specific DNA fingerprints.
SCT40, SCoT45, and SCoT51 are the most informative in determining different types

of mango. 20 types of mangoes are divided into 2 main groups by the ƯPGMA method.
Three indigenous mango varieties including Khodi, Amrutiyo, Kaju, and Dasheri - a

popular mango variety are grouped into one group with a very low similarity of 11 %.

In the clustering pattern, indigenous cultivars - Kaju and Amrutiyo were grouped and
shared 37 % similarity with higher bootstrap values (63 %). This result confirms the

useful application of the SCoT marker system in determining varieties and analyzing

genetic diversity based on their biological characteristics 1'.
In 2015, Fahad Al - Qurainy chose the SCoT primers among various types of DNA
- based polymorphism markers to evaluate the genetic diversity of 6 palm crops in Saudi
Arabia. The results obtained from the polymorphic locus rate (PPL) at the population

level ranged from 3.28 to 13.11 with an average polymorphism value of 7.10. The
genetic diversity of Nei (h) and the Information index of Shannon (I) are 0.033 and 0.046
respectively. However, at the seed level, PPL, the genetic diversity of Nei (h) and the
information index of Shannon (I) are 42.62, 0.090, and 0.1555 respectively. The

ƯPGMA method divided six varieties into five main clusters with a genetic similarity

coefficient of 0.95 l2.

In 2016, Reza Talebi, Farzad Fayaz combined SCoT marker and other DNA -

based polymorphism marker (conserved DNA - derived polymorphism - CDDP) in the

5


Chapter 1. Literature review

study to evaluate genetic diversity and the relationship of Wheat varieties in Iran. 10
CDDP markers and 10 SCoT markers were used to analyze 38 varieties. When
conducting comparisons, the results demonstrated that both the CDDP and SCoT

markers were proved to be more effective than any type of markers and the polymorphic

values of the two markers are relatively similar. The average polymorphic value of
CDDP is 0.39, which is relatively higher than the polymorphic value of SCoT of 0.35.
Using Neighbor - Joining clustering, CDDP, and SCoT markers are used to construct
phylogenetic trees to arrange varieties in three and two main groups respectively. In this

study, CDDP markers have demonstrated more information in studying the genetic
diversity of wheat varieties. The results showed that CDDP and SCoT markers are useful
for analyzing the genetic diversity of wheat varieties 8.

In 2017, Xutian Chai selected the SCoT markers for the study of maximizing
effective sample size in assessing genetic diversity on the subject of vetch. In this study,

they investigated the minimum number of individuals that could represent the genetic

diversity of a single population. Two commercial varieties and two wild varieties were
evaluated using 5 SCoT primers and tested with different amounts of samples: 1, 2, 3,
5, 8, 10,20, 30,40, 50, and 60 individuals. The results showed that the number of alleles

and polymorphic information (PIC) differs between 4 varieties. Cluster analysis by

Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and STRUCTURE

placed the 240 individuals into four distinct clusters. The Expected Heterozygosity (HE)
and PIC increased along with an increase in sampling size from 1 to 10 plants but did

not change significantly when the sample sizes exceeded 10 individuals At least 90 %

of genetic variants in four genotypes was represented when the sample size is 10. Finally,
Xutain Chai at al. concluded that 10 individuals can effectively represent the genetic

diversity of a Vetch population-based on SCoT markers. This study provides theoretical
support for genetic diversity, plant variety identification, evolution, and selection of

varieties that support the identification of common vetch varieties 5.
In 2018, Yan Guo at al recognized the status of Bletilla striata - a rare herbal plant
in China severely affected by excessive exploitation and destruction of natural habitats.

So he and his colleagues conducted a genetic diversity assessment on this subject by

6

Chapter 1. Literature review

combining two molecular markers systems consisting of SCoT and IRAP for exploiting

and utilizing the germplasm resources of Bletilla striata. The result of genetic diversity

across 50 Bletilla striata analyzed by SCoT and IRAP techniques showed that 20 SCoT
primers amplified a total of 209 bands with 201 polymorphisms bands (96.17 %); 8

IRAP primers produced 50 bands with 47 polymorphic bands (94 %). 50 samples of

Bletilla striata were divided into 2 main groups by 2 types of SCoT and IRAP markers
with a genetic similarity coefficient of 0.60 and 0.68 respectively. This showed the rich

genetic diversity among the Bletilla striata varieties from different areas as well as
provided useful information for resource protection work9.

1.3.2 Application of molecular markers in individual identification
There have been studies on the use of molecular markers in individual
identification such as:
SCoT and ISSR molecular markers were used to identify and analyze genetic
comparisons of 23 mango gene samples collected in China's Guangxi province.

Using 18 selective SCoT primers, 158 DNA bands were identified, of which

104 (65.82%) were polymorphic. The eighteen ISSR primers selected amplified

156 bands with 87 (55.77%) polymorphs. 23 varieties were classified into two
main groups based on SCoT analysis and three main groups based on ISSR

analysis with ƯPGMA 6.
Using 10 ISSR primers, from the DNA identification characteristics of 18
samples representing 6 potential avocado strains, based on the presence or

absence of characteristic bands, 9 single-molecule markers, and 25 markers
were identified, double molecule to identify these 06 lines. Provide the

necessary data for the avocado breeding and development in general and

identify the varieties with 6 potential avocado varieties 13.
The M12 has a clear polymorphism between the high sugar and low sugar group.
This SSR can be used to effectively identify high sugar cane varieties 14.

Using 31 SSR (Simple sequence repeats) to identify 382 varieties of cucumber
available in the China market by Target SSR-seq method. Genetic analysis

7


Chapter 1. Literature review

identified four populations: northern China, southern China, European and

Xishuangbanna l5.
The study used 42 superior soybean varieties in Indonesia analyzed with 14
fluorescent SSRs, identified 5 SSRs, namely Satt414, Satt 147, Satt3O8, Satt009,
and Satt516. This 5 SSR can be considered as ID of 42 soybean varieties in
Indonesia l6.

Use the EST - SSR marker set to authenticate the reality of all nine ginseng

plants registered in Korea l7.

8


Chapter 2. Contents and Methods

CHAPTER 2. CONTENTS AND METHODS
2.1 Place of administration
-

Location: Department of Plant Molecular Biology, Nguyen Tat Thanh

University.
-

Duration: 6 months, starting in October 2019.

2.2 Contents
-

Collection of 15 Cat Hoa Loc mango cultivars

-

Extraction of 15 DNA samples

-

Selection and amplification SCoT markers

-

Genetic diversity analyses of 15 Cat Hoa Loc mango cultivars using
NTSYSpc software (version 2.02)

2.3 Methods

2.3.1 DNA extraction
2.3.1.1 Collection of leaf samples
Sample collection location: Samples were collected in Southern Vietnam,

including 2 samples from Ben Tre, 9 samples from Tien Giang, 1 sample from Long An,

3 samples from Khanh Hoa, and 15 samples from Southern Horticultural Research
Institute (SOFRI).
Leaves sample: Leaves samples should be the 4th or 5th one from the top of the

branches.

Preliminary treatment of collected leaf samples: Leaves samples were washed
underwater. 70 % alcohol was used to wipe the leaves, which were then left in indoor
conditions to make them dry.

Samples preservation if not immediately extracted: the cleaned leaves samples
were placed into zip bags and labeled the sampling date and place, then stored in the

freezer at - 21 °C

9

Chapter 2. Contents and Methods

2.3.1.2 Total DNA extraction
To weigh the sample, the foil was put on the analytical balance, the scissors and

tweezers that have been disinfected were used to cut the leaf sample, each leaf samples
had an approximate weight of 100 mg. Then the leaf samples were separated into small

pieces and placed in 2.0 ml tubes to prepare for DNA extraction.
The method of total DNA extraction (according to ABT kit code HI - 123) includes

the following steps:
The liquid nitrogen was put into 2.0 ml tubes that already contained leaf sample,

the samples were then crushed with a glass rod until the samples were broken into a fine
powder.

Next, the extraction process was carried out according to the protocol attached to
the manufacturer's ABT Kit, including:
Step 1: Sample resolution:

-

400 pl PLS 1 solution was added into 2 ml tube (containing the sample to

extract) and then vortexed evenly.

-

10 pl Rnase was added, then lighten vortexed and incubated at 65 °C for 30
minutes until the cell is completely dissolved.

-

130 pl PLS 2 was added, then mixed completely and incubated for 5 minutes
on ice at 4 °C.

-

After incubation, tubes were centrifuged at maximum speed (15000 rpm) for 5
minutes and all floating fluids (about 450 pl) were sucked into 1.5 ml tube.

Step 2: DNA Attachment to the silica column:

-

675 pl PBB solution was added and stirred evenly at room temperature for 1 2 minutes.

-

Then, 600 pl floating fluid after adding PBB was transferred into the silica

column.

-

Silica columns were centrifuged at 11000 rpm.

-

The filtrates were removed, the silicas containing the solution were reused and

retained.

10


Chapter 2. Contents and Methods

-

Next, the silicas were centrifuged again with the amount of remaining mixture.

Step 3: Wash the column for the first time

-

500 pl PWB was transferred into the silica column.

-

The silicas were centrifuged at 11000 rpm for 1 minute.

-

The filtrates were removed, the silicas containing the solution were reused and

retained.

Step 5: Wash the column for the second time

-

500 pl PWB was transferred into the silica column.

-

The silicas were centrifuged at 11000 rpm for 1 minute.

-

The filtrates were removed, the silicas containing the solution were reused and

retained.
Step 6: Dry silica column

-

The silicas were centrifuged at 11000 rpm for 2 minutes.

-

The solution in the tubes was removed and silica columns were retained.

Step 7: DNA storage

-

Silica columns were transferred into 1.5 ml tube.

-

100 pl EB solution (incubated at 70 °C) was added to the silica column.

-

Centrifugal 11000 rounds in 2 minutes

Keep 1.5 ml tube and store - 20 °C if not used.

2.3.1.2 DNA quality testing
Qualitative testing of total DNA samples was carried out by the electrophoresis
method on 0.8 % standard agarose gel with a 6X red gel dye color. Results were shown
on electrophoresis gel with the appearance of bright bands indicating total DNA.

Quantify total DNA by OD system: Total DNA quantitative reactions were
conducted with 3 pl per reaction.

11


Chapter 2. Contents and Methods

2.3.2 Screening polymorphic SCoT markers
All of forty - six SCoT markers (Table 2.1) were utilized from researches of Luo 4,
Gajera ", Yan 9, and Yong 7 and purchased from PHUSA Biochem Co., Ltd.

Polymorphism Screening of SCoT markers with 15 Cat Hoa Loc mango cultivars were
carried out by PCR reaction 3 times for uniform thermal cycles (Table 2.2) and

components (Table 2.3). The DNA bands were scored only for clear and reproducible

ones which were not detected any new bands after 3 repeats.

Table 2.1 Sequences of SCoT markers using in this study 4’7’9’11
Primer

Sequence (5' to 3')

ID

Cite

Primer

from

ID

Sequence (5' to 3')

Cite

from

SCoTOl

CAACAATGGCTACCACCA

Luo

SCoT26

ACCATGGCTACCACCGTC

Gajera

SCoT02

CAACAATGGCTACCACCC

Gajera

SC0T27

ACCATGGCTACCACCGTG

Yong

SC0TO3

CAACAATGGCTACCACCG

Luo

SCoT28

CCATGGCTACCACCGCCA

Yong

SCoT04

CAACAATGGCTACCACCT

Yan

SCoT30

CCATGGCTACCACCGGCG

Yan

SCoT05

CAACAATGGCTACCACGA

Gajera

SCoT31

CCATGGCTACCACCGCCT

Yong

SC0TO6

CAACAATGGCTACCACGC

Gajera

SCoT32

CCATGGCTACCACCGCAC

New

SCoT07

CAACAATGGCTACCACGG

Yan

SC0T33

CCATGGCTACCACCGCAG

Gajera

SC0TO8

CAACAATGGCTACCACGT

Yong

SC0T34

ACCATGGCTACCACCGCA

Luo

SCoT09

CAACAATGGCTACCAGCA

Luo

SC0T35

CATGGCTACCACCGGCCC

Yong

SCoTIO

CAACAATGGCTACCAGCC

Yan

SCoT36

GCAACAATGGCTACCACC

New

SCoTll

AAGCAATGGCTACCACCA

Yong

SCoT40

CAATGGCTACCACTACAG

Gajera

SC0TI2

ACGACATGGCGACCAACG

Yong

SC0T44

CAATGGCTACCATTAGCC

New

SCoT13

ACGACATGGCGACCATCG

Yan

SC0T45

ACAATGGCTACCACTGAC

Gajera

SCoT14

ACGACATGGCGACCACGC

Gajera

SCoT51

ACAATGGCTACCACTGTC

Gajera

SCoT15

ACGACATGGCGACCGCGA

Yong

SC0T6O

ACAATGGCTACCACCACA

New

SC0TI6

ACCATGGCTACCACCGAC

Gajera

SC0T6I

CAACAATGGCTACCACCG

Luo

SCoT17

ACCATGGCTACCACCGAG

Yong

SC0T63

ACCATGGCTACCACGGGC

Gajera

SC0TI8

ACCATGGCTACCACCGCC

Yan

SC0T65

ACCATGGCTACCACGGCA

Gajera

SCoT19

ACCATGGCTACCACCGGC

Gajera

SC0T66

ACCATGGCTACCAGCGAG

Gajera

SCoT20

ACCATGGCTACCACCGCG

Luo

SCoT70

ACCATGGCTACCAGCGCG

Gajera

SC0T22

AACCATGGCTACCACCAC

Yong

SC0T73

CCATGGCTACCACCGGCT

Gajera

SC0T23

CACCATGGCTACCACCAG

Yong

SC0T77

CCATGGCTACCACTACCC

Gajera

SC0T25

ACCATGGCTACCACCGGG

Luo

SCoT78

CCATGGCTACCACTAGCA

Gajera

12


Chapter 2. Contents and Methods

Table 2.2 Component content of PCR reaction 11
No.

Components

Volumn

1

DNA Tag mix 2X

6 pl

2

SCoT primers

Ipl

3

DNA sample

2 pl

4

Water (ddH2O)

3 pl

Total volumn

12 pl

Table 2.3 PCR cycling conditions 4’11
Step

Tempetature (°C)

Time

Cycles

Initial denaturation

94

3 mins

1

Denaturation

94

45 secs

Annealing

50

1 mins

Extension

72

2 mins

Final extension

72

5 mins

40

1

Electrophoresis method: PCR products were detected by electrophoresis
on agarose gel with a concentration of 0.8 %.
Step 1: Prepare 0.8 % agarose gel

- Add 0.4 g of agarose powder to Erlen, add 50 ml of TBE, shake to dissolve.
- Put in microwave for 1 - 2 minutes.
- Allow to cool to 50 ° c then pour into a pre-set mold.
- Let stand for 15-20 minutes for the gel to solidify.
Step 2: Inject the sample into the gel well and run the electrophoresis

- Put the prepared gel pad into the electrophoresis tank containing the TBE solution,
the well of the gel pad is facing the cathode.

- Add 2 pl of gelred 6X and 12 pl of PCR sample then mix well.
- Pump all 14 pl of the mixture into the gel well.
13


Chapter 2. Contents and Methods

- Inject the DNA ladder into the first well.
- Close the lid of the electrophoresis tank and plug in the power source.
- Electrophoresis is performed for 90 minutes at 45 V.
Step 3: Check the results of electrophoresis

- After electrophoresis, place the gel into the gel scanner.
- Observe and photograph the results.

2.3.3 PCR data analysis

2.3.3.1 Input data
After electrophoresis and reading the results on the gel, enter data obtained from
on agarose gel as 0 and 1 representing of absence and presence of a DNA band in the
following order in Microsoft Office Excel 97 ~ 2016:

-

Enter each primer’s data in a separate data sheet in a single file.

-

Write always “1” in “Al” cell as a sign of a rectangular matrix.

-

In “Bl” cell write the total number of individuals (isolates or varieties or races
or genotypes, etc.).

-

In “C1 ” cell write the maximum band number you have got using this primer.

Leave blank the second raw (A2, B2, C2.Z2).

-

Write the name, number, code, or acronym of individuals starting from “A3” ~

“An”, n = number of - individuals.

-

Enter 0 and 1 data in “B3” ~ “Z3” for the first individual and “B4” ~ “Z4” for
the second one and ...

-

Save the file as an arbitrary name using by “Save as” sub-menu in File menu l8.

-

After that, synthesis and import all primers data into a separate datasheet.

14


Chapter 2. Contents and Methods

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23.3.2 Data accession and clustering analysis
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Figure 2.2 Example of similarity matrice of sample
With NTSYSpc 2.02, we could access the similarity matrice by the “Similarity”
tab and draw the dendrogram by “Clustering” tab 18.
15


Chapter 2. Contents and Methods

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Figure 2.3 Example of dendrograms drawing (phylogenetic trees)

16


Chapter 3. Results and Discussion

CHAPTER 3. RESULTS AND DISCUSSION
3.1 Sample collection
Fresh leaves of 15 Cat Hoa Loc mango samples were collected in Southern

Vietnam, including 2 samples from Ben Tre, 9 samples from Tien Giang, 1 sample from
Long An, and 3 samples from Khanh Hoa. In addition, 15 samples were also collected

from Southern Horticultural Research Institute (SOFRI), Long Dinh Village, Chau
Thanh District, Tien Giang Province, and were coded from Al to A15 (Table 3.1).

Table 3.1 List of mango cultivars used in this study
No.

Name of sample

Code

1

Cat Hoa Loc 1

XI

Collecting location
Hoa Loc Village, Cai Be District, Tien Giang
Province

Hoa Loc Village, Cai Be District, Tien Giang
2

Cat Hoa Loc 2

X2
Province

Hoa Loc Village, Cai Be District, Tien Giang

3

Cat Hoa Loc 3

X3
Province

Hoa Loc Village, Cai Be District, Tien Giang
4

Cat Hoa Loc 4

X4
Province

Hoa Loc Village, Cai Be District, Tien Giang
5

Cat Hoa Loc 5

X5
Province

Hoa Loc Village, Cai Be District, Tien Giang
6

Cat Hoa Loc 6

X6
Province

Southern Horticultural Research Institute
7

Cat Hoa Loc 7

X7

(SOFRI), Long Dinh Village, Chau Thanh

District, Tien Giang Province

Southern Horticultural Research Institute
8

Cat Hoa Loc 8

X8

(SOFRI), Long Dinh Village, Chau Thanh

District, Tien Giang Province

17