VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ------- ------- GRADUATION THESIS PROJECT: EVALUATION OF GENETIC DIVERSITY AND PRESENCE OF LATE BLIGHT (PHYTOPHTHORA INFESTANS) RESISTANCE GENES IN SOME PROMISING HYBRID POTATO LINES BY MOLECULAR MARKERS HA NOI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ------- ------- GRADUATION THESIS PROJECT: EVALUATION OF GENETIC DIVERSITY AND PRESENCE OF LATE BLIGHT (PHYTOPHTHORA INFESTANS) RESISTANCE GENES IN SOME PROMISING HYBRID POTATO LINES BY MOLECULAR MARKERS Student Name : Le Van Ngoc Class : K63CNSHE Student Code : 637424 Instructor : Nguyen Xuan Truong, Ph.D HA NOI – 2022 COMMITMENT I assure that the whole research process was performed by myself under the scientific guidance of Ph. Nguyen Xuan Truong. I assure that all research contents, results, and information in my thesis is completely honest and unpublished. Hanoi, December 05th 2022 Student Le Van Ngoc i ACKNOWLEDGEMENTS In order to complete the graduation thesis, besides my own efforts, I have received a lot of enthusiastic support from teachers, friends and relatives.
First, I would like to start by expressing my sincere gratitude to Nguyen Xuan Truong, a PhD. supervisor for helping me finish my thesis with their guidance and expertise. Additionally, their advice was helpful to me as I was preparing this thesis. I want to thank PhD.
Dinh Truong Son and all my teachers in the Faculty of Biotechnology, especially the Department of Plant Biotechnology. They provided useful scientific knowledge as well as technique for me and where supporting equipment and finances for my thesis. One of the most crucial elements that will enable me to successfully complete my thesis is this. I appreciate the passionate assistance from the Institute of Biotechnology and everyone who works there so that I can have the finest thesis.
I thank my lab-mate for his meaningful contribution. I will appreciate the time when we were working together. Last but not least, I would specifically like to thank my family, especially my parents who have loved me, cared for me, trusted me and given me the best study condition. Hanoi, December 05th 2022 Student Le Van Ngoc ii CONTENTS COMMITMENT .v LIST OF TABLES.
Purposes and requirements. Introduction of potato. Nutrient content in potatoes. Potato production in the world.
Potato production in Vietnam. Phytophthora infestans in potato. The cause to the illness. Molecular markers and applications of RAPD and ISSR markers in genetic diversity analysis.
RAPD molecular marker. ISSR molecular marker .4 Researches on potato (Solanum tuberosum L.) using RAPD and ISSR molecular markers. MATERIALS AND METHODS. Time and Place of the Study.
RESULTS AND DISCUSSION. Genetic diversity analysis of 28 potato lines using RAPD molecular. Genetic diversity analysis using ISSR molecular markers. Genetic diversity analysis combined RAPD and ISSR molecular markers.
CONCLUSION AND SUGGESTION .44 iv ABBREVIATION CTAB Cetyl trimethylammonium bromide DNA Deoxyribonucleic Acid dNTPs Deoxynucleotide Solution Mix EDTA Etylen Diamine Tetra Acetic Acid ISSR Inter-Simple Sequence Repeats RAPD Random Amplified Phlymorphic DNA PCR Polymerase chain reaction TAE Tris base, acetic acid and EDTA PVPP Poly Vinyl Poly Pyrrolidone v LIST OF TABLES Table 2.1 Potato yield of the top 5 countries in the world in 2012 and 2013 .1: 48 potato lines and three commercial varieties used for the study .2 List of chemical substances used in research .3 List of RAPD primers used in research .4 List of ISSR primers used in research .5 List of molecular markers associated with late blight resistance genes in potato .6 Components of each PCR reaction. Thermal cycler of PCR. Concentration and quality of DNA isolated from 48 potato line and varieties .2 The present of resistant gene in studied potato lines, symbol “+” for presence the gene, empty cells represent no detection.3 Polymorphism of 28 lines of potato revealed by RAPD markers .4 Matrix of genetic similarity among 28 accessions of potato revealed by RAPD markers calculated by similarity coefficient of Sokal-Michener 30Error! Bookmark not defined. Polymorphism of 28 potato lines accessions revealed by ISSR markers.6 Matrix of genetic similarity among 28 potato accessions revealed by ISSR markers calculated by similarity coefficient of Sokal-Michener .7 Matrix of genetic similarity among 28 potato accessions revealed by combined RAPD and ISSR markers calculated by similarity coefficient of Sokal-Michener .8 Matrix comparisons of Mantel test / Two-tailed test between markers .41 vi LIST OF FIGURES Figure 2.
Botanical characteristics of potato. Results of electrophoresis product with primer APG-05. UPGMA cluster analysis of 28 accessions of potato lines with a similarity coefficient of RAPD marker. Principal component analysis (PCA) of 28 potato accessions based on RAPD marker.
UPGMA cluster analysis of 28 potato accessions with a similarity coefficient of ISSR marker. PCA analysis of 28 potato accessions based on ISSR markers. UPGMA cluster analysis of 28 potato accessions with a similarity coefficient combined RAPD and ISSR marker. Principal component analysis (PCA) of 28 potato accessions based on pooled RAPD and ISSR marker.
Preface Potato (Solanum tuberosum L.) is a short-term crop that plays a significant role in human food crops. It is a member of the Solanaceae family and is native to Bolivia and Peru’s Andes mountains. In terms of food production, potatoes are currently the fourth most widely planted crop after rice, wheat and maize. They are the most widely grown tuber in the world.
They can adapt to a variety of environment situations, are simple to grow and produce a speedy harvest (International Year of the Potato 2008; Jeff Chapman et al. The productivity and quality of tubers are, however, greatly impacted by a number of illnesses that are frequently transmitted to potatoes. Diseases on potatoes are very diverse, rich in composition and cause of disease such as late blight, yellow wilt, viral leaf curl… Late blight caused by Phytophthora infestans is considered the most harmful in the world, resulting approximately 16% output losses globally (Haverkort A et al. With the strong development of molecular biology, the breeding technology of molecular markers ensure accuracy, and be able to gather many targeted genes into one variety.
Traditional breeding methods are difficult or impossible to do. To create a new potato variety, the first thing is to have a source of resistance genes and molecular markers associated with those resistance genes, then evaluate and determine the resistance to late blight of the variety to use for future breeding. Therefore, I conduct this work: “Evaluation of genetic diversity and presence of late blight (Phytophthora infestans) resistance genes in some promising hybrid potato lines by molecular markers”. Purposes and requirements 1.
Purposes - Finding promising potato lines containing late blight resistance genes. - Besides, Analysis the genetic diversity and relationships between 28 potato samples by RAPD and ISSR molecular markers. Requirements - Using DNA molecular markers to detect resistance genes in potato seed samples - Evaluation of the genetic diversity of the potato seed samples 2 PART 2. Introduction of potato 2.
Origin Potato (Solanumtuberosum L.) is indigenous to the highlands of South America’s Andes Mountains, where it grows at elevations of 2000-5000 meters. When the Spaniards first arrived in the Magdalena valley (South America), they found potatoes, beans and grain there along with the Indians who had fled. Due to the vibrant blossoms at the time, people nicknamed potatoes truffles (Salaman, 1949). Numerous historical locations have been uncovered by scientists as evidence that the potato plant existed as far back as 500 BC.
Farmers cultivated hundreds of different varieties of potatoes throughout the mountains of what are now Bolivia, Chile, Colombia, Ecuador, and Peru during the Spanish conquest of the Americas in the 16th century (Horton, 1987). The original potato types are still grown by the Indians of the Titicaca region today (Ducreux, 1989). A Frenchman who serves as the head of the Hanoi Botanical Garden is responsible for bringing potatoes to our nation. We called this plant "potato" because the French introduced it to our nation and made its cultivation popular (Vu Huong Van, 2007).
At the moment, potato cultivation is concentrated in the Red River Delta, Sapa, and Da Lat, all of which have cold and temperate climates. Genetics Worldwide, there are around 5,000 different potato kinds. The Andes alone contain 3,000 of them, mostly in Peru, Bolivia, Ecuador, Chile, and Colombia. Depending on the taxonomic school, they belong to either eight or nine species.
There are more than 200 wild species and subspecies in addition to the 5,000 cultivated cultivars, many of which can interbreed. Numerous crossbreeding attempts have been made to add pest and disease resistances from the gene pool of wild species to the gene pool of domesticated potato species. 3 Modern forms of Solanum tuberosum, a tetraploid species with 48 chromosomes, are the most frequently grown plant in the world. The four other species are S.
ajanhuiri, all of which are diploid (have 24 chromosomes). The two species that are triploid (with 36 chromosomes each) are S. One cultivable pentaploid species, S. curtilobum, has 60 chromosomes.
Solanum tuberosum has two main subspecies: andigena, often known as Andean, and tuberosum, also known as Chilean. The Chilean potato, a native of the Chiloé Archipelago, is adapted to the long-day conditions common in the higher latitude region of southern Chile, unlike the Andean potato, which is adapted to the short-day conditions prevalent in the mountainous equatorial and tropical regions where it originated. There are 4,870 different varieties of potato germplasm available at the International Potato Center in Lima, Peru, the majority of which are conventional landrace cultivars. The potato genome is a medium-sized plant genome with 12 chromosomes and 860 million base pairs, according to the 2009 announcement by the international Potato Genome Sequencing Consortium.
A subspecies that formerly flourished in the lowlands of south-central Chile is directly ancestors of more than 99 percent of the potato types farmed today. But genetic analysis of the several cultivars and wild species confirms that all potato subspecies originate from a single origin in the region of modern-day southern Peru and extreme Northwestern Bolivia (from a species in the Solanum brevicaule complex). Although at least one wild potato species, Solanum fendleri, naturally ranges from Peru into Texas, where it is used in breeding for resistance to a nematode species that attacks cultivated potatoes, the majority of modern potatoes grown in North America arrived through European settlement and not independently from the sources in South America. The hexaploid Solanum demissum, a source of resistance to the deadly late blight disease, is one of the significant wild species that have been heavily employed in modern breeding.
Mexico is a secondary center of genetic variety for potatoes. The potato has been genetically modified to withstand potato blight using Solanum bulbocastanum, a different cousin that is endemic to this area. Botanical characteristics When potatoes are produced from seed tubers, only bunch roots form; when potatoes are grown from seeds, tap roots form; from tap roots, numerous auxiliary roots form. Although the roots of the underground stems, commonly referred to as tubers, are likewise capable of growing, they are shorter and less branching.
The root system has roots that extend up to 1.5 to 2 meters below the surface of arable soil, where it is primarily dispersed (Ta Thu Cuc, 2007). Potato stems typically grow straight, occasionally zigzag, have three to four sides, and range in height from 40 to 70 cm to 1 to 1. The amount of time needed to maintain a plant's height might vary depending on the species. Depending on the cultivar, the stem is often green, light green, dark green, or occasionally pink or purple in color.
The size of the leaf plate, the compound leaves' bifurcation, the number of pairs growing symmetrically across the axis, and the size of the odd leaf at the top of the plant, known as the apical leaflet, all depend on the variety, season, and care conditions of the plant.