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

UNIVERSITY True learning – True practice – True success – True future FACULTY OF BIOTECHNOLOGY GRADUATION THESIS APPLICATION OF MOLECULAR MARKER: START CODON TARGET SCOT IN INDIVIDU

UNIVERSITY

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

NGUYEN TAT THANH UNIVERSITY

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

Major : Biotechnology Supervisor : MSc Nguyen Thi Nha

Ho Chi Minh City, 2020

- -oOo -

ASSIGNED TASK OF GRADUATION THESIS

1 Thesis’s title:

Application of molecular marker: Start Codon Target (SCoT) in individual

identification of Cat Hoa Loc mango (Mangifera indica L)

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 Loc mango

3 Contents:

- Collection of 15 Cat Hoa Loc mango cultivars

- Extraction of 15 DNA samples

- Selection and amplification of SCoT markers with DNA samples

- Genetic diversity analysis of 15 Cat Hoa Loc mango cultivars using NTSYSpc software

4 Execution time: from October 2019 to July 2020

5 Supervisor: MSc Nguyen Thi Nha

Contents and requirements of this thesis were adopted by the subject

TP Ho Chi Minh City, 7 th July, 2020

ACKNOWLEDGEMENTS

I would like to express my special thanks of gratitude to my supervisor MSc

Nguyen Thi Nha as well as all the teachers who gave me the golden opportunity to

do this thesis on the topic “Application of molecular marker: Start Codon Target

(SCoT) in individual identification of Cat Hoa Loc mango (Mangifera indica L)”,

which also helped me in doing a lot of experiments and I came to know about so many

new things I am thankful to them

Secondly, I would also like to thank my parents and friends in my lab who

helped me a lot in finalizing this thesis within the limited time frame

Pham Thi Hoa Mai Faculty of Biotechnology Nguyen Tat Thanh University

TABLE OF CONTENTS

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 of SCoT directive application 4

1.3.2 Application of molecular markers in individual identification 7

2.1 Place of administration 9

2.2 Contents 9

2.3 Methods 9

2.3.1 DNA extraction 9

2.3.2 Screening polymorphic SCoT markers 12

2.3.3 PCR data analysis 14

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

ABSTRACT

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

individual identification of Cat Hoa Loc mango (Mangifera indica L)” 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

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 SCoT06, SCoT14, SCoT18, and SCoT44 across 15 Cat Hoa Loc mango cultivars 23

Figure 3.3 Similarity matrix of 27 polymorphic SCoT marker across 15 Hoa Loc 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 SOFRI 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

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

LIST OF ACRONYMS

SCoT Start Codon Target

UPGMA Unweighted Pair Group Method with Arithmetic mean

PLS Plant Lysis buffer

PBB Plant Binding buffer

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 (Mangifera 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

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 Loc mango

LITERATURE REVIEW

1.1 Introduction of Cat Hoa Loc Mango tree

Scientific name and location of Mango in the classification system:

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-to-fruit process from, good drought tolerance 1

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

Figure 1 Picture of Cat Hoa

Loc mango

Chapter 1 Literature review

1.1.2 Distribution place

Vietnam is a long-standing mango-growing country and mango is grown in various different ecological regions, but the distribution is still mainly in southern provinces Tien Giang province now has over 1,579 ha in areas planting Hoa Loc mango, which mainly concentrated in areas along Tien river in Cai Be and Nam Cai Lay districts such

as Hoa Hung, An Huu, An Thai Trung, Tan Hung, ., annual output is estimated at 35,926 tons, yield 23 tons/ha In Tien Giang, Cai Be district is the largest area planting mango at 3,622 ha, accounting for 52 % of the total mango planting area in the province Hoa Loc mango planting area in Hau Giang is about 5,500 ha concentrated in Chau Thanh, Chau Thanh A, Phung Hiep, Vi Thuy and Long My districts, Nga Bay town, and

Vi Thanh City

Dong Thap is the province with the largest mango planting area in the Mekong Delta with more than 9,300 ha, of which Hoa Loc mango accounts for 20 % of the area 2

1.1.3 The values of Cat Hoa Loc mango

Nutritionally, Hoa Loc Mango ranks in the top 5 among the nutritious fruits Mango minerals involve calories, lipids, cholesterol, Sodium 1, Potassium 168, carbohydrates, fiber, sugar, protein, calcium, iron, vitamin C, vitamin B1, vitamin B6, magnesium, etc

Cat Hoa Loc mango also works in:

- Cancer prevention: The oxidants that present in mangoes such as isoquercitrin, fisetin, quercetin, methyl gallate, astragalin, gallic acid, ect are effectively useful for helping the body fighting against the formation and growth of cancer cells Studies show that the antioxidants in mangoes prevent breast cancer, colon cancer, and prostate cancer

- Strengthen resistance: Hoa Loc mango is a good source of vitamin C, vitamin A and various carotenoids that help strengthen the immune system

- Digestive support: Mango contains enzymes that help break down proteins, facilitate the absorption into the body, the fiber in mango also helps a lot in supporting the digestive system to work

- 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 phylogenetics 3

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 plants 4

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 plants 6

The SCoT markers have been successfully implemented in analyzing genetic diversity in many subjects including mango 4, sugar cane 7, wheat 8, medicinal plants 9 , 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 10

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

similarity coefficient at 0.674 High genetic diversity was found on three main sugarcane cultivars including ROC22, ROC16, and ROC10 (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 UPGMA 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 11

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 UPGMA method divided six varieties into five main clusters with a genetic similarity coefficient of 0.95 12

In 2016, Reza Talebi, Farzad Fayaz combined SCoT marker and other DNA – based polymorphism marker (conserved DNA – derived polymorphism – CDDP) in the

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

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 work 9

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 UPGMA 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

Chapter 1 Literature review

identified four populations: northern China, southern China, European and Xishuangbanna 15

- The study used 42 superior soybean varieties in Indonesia analyzed with 14 fluorescent SSRs, identified 5 SSRs, namely Satt414, Satt147, Satt308, Satt009, and Satt516 This 5 SSR can be considered as ID of 42 soybean varieties in Indonesia 16

- Use the EST - SSR marker set to authenticate the reality of all nine ginseng plants registered in Korea 17

CONTENTS AND METHODS

- 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

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 µl PLS 1 solution was added into 2 ml tube (containing the sample to extract) and then vortexed evenly

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

- 130 µl 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 µl) were sucked into 1.5 ml tube Step 2: DNA Attachment to the silica column:

- 675 µl PBB solution was added and stirred evenly at room temperature for 1 –

2 minutes

- Then, 600 µl 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

- Next, the silicas were centrifuged again with the amount of remaining mixture Step 3: Wash the column for the first time

- 500 µl 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 µl 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 µl 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 µl per reaction

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 11, 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

SCoT03 CAACAATGGCTACCACCG Luo SCoT28 CCATGGCTACCACCGCCA Yong

SCoT04 CAACAATGGCTACCACCT Yan SCoT30 CCATGGCTACCACCGGCG Yan

SCoT05 CAACAATGGCTACCACGA Gajera SCoT31 CCATGGCTACCACCGCCT Yong

SCoT06 CAACAATGGCTACCACGC Gajera SCoT32 CCATGGCTACCACCGCAC New

SCoT07 CAACAATGGCTACCACGG Yan SCoT33 CCATGGCTACCACCGCAG Gajera SCoT08 CAACAATGGCTACCACGT Yong SCoT34 ACCATGGCTACCACCGCA Luo

SCoT09 CAACAATGGCTACCAGCA Luo SCoT35 CATGGCTACCACCGGCCC Yong

SCoT10 CAACAATGGCTACCAGCC Yan SCoT36 GCAACAATGGCTACCACC New

SCoT11 AAGCAATGGCTACCACCA Yong SCoT40 CAATGGCTACCACTACAG Gajera SCoT12 ACGACATGGCGACCAACG Yong SCoT44 CAATGGCTACCATTAGCC New

SCoT13 ACGACATGGCGACCATCG Yan SCoT45 ACAATGGCTACCACTGAC Gajera SCoT14 ACGACATGGCGACCACGC Gajera SCoT51 ACAATGGCTACCACTGTC Gajera SCoT15 ACGACATGGCGACCGCGA Yong SCoT60 ACAATGGCTACCACCACA New

SCoT16 ACCATGGCTACCACCGAC Gajera SCoT61 CAACAATGGCTACCACCG Luo

SCoT17 ACCATGGCTACCACCGAG Yong SCoT63 ACCATGGCTACCACGGGC Gajera SCoT18 ACCATGGCTACCACCGCC Yan SCoT65 ACCATGGCTACCACGGCA Gajera SCoT19 ACCATGGCTACCACCGGC Gajera SCoT66 ACCATGGCTACCAGCGAG Gajera SCoT20 ACCATGGCTACCACCGCG Luo SCoT70 ACCATGGCTACCAGCGCG Gajera SCoT22 AACCATGGCTACCACCAC Yong SCoT73 CCATGGCTACCACCGGCT Gajera SCoT23 CACCATGGCTACCACCAG Yong SCoT77 CCATGGCTACCACTACCC Gajera SCoT25 ACCATGGCTACCACCGGG Luo SCoT78 CCATGGCTACCACTAGCA Gajera