VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY --------� � � -------- UNDERGRADUATE THESIS TITLE: SCREENING OF POTATO GERMPLASM FOR FLOWERING AND SELF-INCOMPATIBILITY Hanoi-2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY --------� � � -------- UNDERGRADUATE THESIS TITLE: SCREENING OF POTATO GERMPLASM FOR FLOWERING AND SELF-INCOMPATIBILITY Practicing student’s name : TRAN THI TRANG Student's code : 620643 Class : K62CNSHE Faculty : BIOTECHNOLOGY Supervisor : PHANHUU TON, PROF. Hanoi - 2022 COMMITMENT I hereby declare that the data and research results in this thesis are true and do not copy the results of any previous undergraduate reports. Undergraduation thesis with references to documents, citation information is indicated in the references section. Hanoi, March 9th, 2022 Sincerely, Tran Thi Trang i ACKNOWLEDGEMENT In order to complete the undergraduation thesis, in addition to my own efforts, I have received the great attention and support of teachers, friends as well as family members.
First of all, I would like to express my deep gratitude to PhanHuu Ton, Prof., who enthusiastically guided, oriented, and helped me professionally throughout the process of completing my graduation thesis. Secondly, I would like to thank Engineers PhanThanh Tung, PhanHuu Hien and PhamDinh On work at the Center for Conservation and Development of Crop genetic resources and all teachers in the Department of Molecular Biology and Applied Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture, are very enthusiastic to help, create the most favorable conditions for me during my internship at the center and the subject. I would also like to thank the teachers of the Vietnam National University of Agriculture for equipping me with the necessary knowledge over the past 4 years so that I could conduct and complete my undergraduation thesis. Finally, I would like to sincerely thank my family members, brothers and sisters, friends who have encouraged, helped and created conditions for me to complete this undergraduation thesis.
ii CONTENTS COMMITMENT. iii LIST OF TABLES. v LIST OF FIGURES AND GRAPHS .vi LIST OF ABBREVIATIONS. OVERVIEW OF POTATO.
Origin and classification of potato crop. Botanical properties of the potato crop. Biological characteristics of potato crop. Genetics and reproductive characteristics of potatoes.
Some factors affect the growth and development of potato crop. Nutritional requirements of potato crop. Role in the World Food Supply. Time to plant potatoes in Vietnam.
The situation of potato production in the world. Potato production situation in Vietnam. Self-incompatibility gene. MATERIAL AND METHOD.
Location and time. Evaluation method of some agro-biological characteristics. PCR method to identify self-incompatibility genes (S11 and S16) (Olga Marcellan et al, 2012). Evaluation of flowering.
RESULTS AND DISCUSSION. Evaluation results of some agro-biological characteristics. Results of monitoring some indicators of growth and development characteristics. Results of monitoring a number of indicators on yield and quality of tubers.
Results of PCR method to identify self-incompatibility genes (S11 and S16). DNA extraction results. Results of evaluating the flowering ability of the experimental varieties. CONCLUSION AND SUGGESTION.
49 iv LIST OF TABLES Table 2. Representative potato genetic markers and maps. List of screening materials. DNA molecular markers used in the experiment.
Growth time tracking table. Monitor stem and leaf characteristics. Yield survey data of potato varieties. Quality monitoring table of tubers.
PCR results to detect S11 and S16 self-incompatibility genes. 43 v LIST OF FIGURES AND GRAPHS Figure 2. Parts of the potato plant. Results of total DNA electrophoresis of 40 potato varieties.
Electrophoresis results of PCR products detecting self- incompatibility genes S11 and S16. Flower images represent a number of varieties with flowering. Image of tubers of high yielding potato varieties. 46 vi LIST OF ABBREVIATIONS AFLP: amplified fragment length polymorphism CTAB: cetyltrimethylammonium bromide DArT: diversity array technology DNA: deoxyribonucleic acid EDTA: etylenediaminetetraacetic acid GSI: gametophytic self-incompatibility ISSR: inter simple sequence repeat PCR: polymerase chain reaction RAPD: random amplified polymorphism RFLP: restriction fragment length polymorphism RGL: resistance gene-like fragment SI: Self-incompatibility SLF: S-locus F-box SNP: single nucleotide polymorphism S-RNase: S-Ribonucleases SSCP: single stranded conformation polymorphism SSR: simple sequence repeat, microsatellite TE: Tris-EDTA vii Chapter 1.
Introduction Potato, Solanum tuberosum, belongs to the family Solanaceae, native to the Andes mountains of Bolivia and Peru, is a short-term crop, occupying an important role in food crops for people. Currently, potatoes are the world's most widely grown as root crop and the fourth most popular crop in terms of yield - behind rice, wheat and maize.Potatoes have the characteristics of being easy to grow, giving a short harvest and adapting to different environmental conditions. Potato production has many forms such as: asexual propagation, sexual propagation, tissue culture. In which, sexual propagation is a method of propagation by seeds, but this method is rarely used because in potatoes there is a phenomenon of self-incompatibility which hinders inbreeding.
Self-incompatibility (SI) is common in tuber-bearing Solanum species, especially at the diploid level. In most diploid potato genotypes, inbreeding is severely limited by a gametophytic self-incompatibility (GSI) system that is controlled by the multi-allelic S-locus (16 alleles name as S1, S2, .S16), each allele codes for one S-RNase (S-Ribonucleases) and several S-locus F-box (SLF) with different pollen-specific traits, the S locus location was identified on chromosome I. Molecularly, self-incompatibility is due to the cytotoxic effect of S-RNases that inhibit pollen tube growth preventing fertilization. In order to serve potato breeding by seed, it is necessary to investigate the potatoes germplasm for some agro-biological characteristics and self- incompatibility alleles using PCR-based DNA molecular markers.
Currently, the Center for Conservation and Development of Crop Genetic Resources has collected a potatoes germplasm, which is a diverse genetic resource for the study of self-incompatibility genes for potato breeding by seed. So with the guidance of PhanHuu Ton Prof., we conduct the title: "Screening of potatoes germplasm for flowering and self-incompatibility". Purpose Evaluation of some agro-biological characteristics, determining the ability flowering and containing genes of self-incompatibility (S11 and S16) of the studied potato varieties. Requirement Evaluation of some agro-biological characteristics affecting the growth- development, yield and quality of the studied potato varieties.
Determining the ability to contain self-incompatibility genes (S11 and S16) of the studied potato varieties. Evaluation of flowering ability of studied potato varieties. OVERVIEW OF POTATO 2. General introduction Potatoes (Solanum tuberosum), belonging to the Solanaceae family, are grown for their starchy tubers.
They are the world's most widely grown root crop and the fourth most popular crop in terms of fresh yield - behind rice, wheat and maize. Potato is one of the crops with high economic efficiency because of its short growing time (80-100 days), easy to grow, high yield in mild climate conditions with suitable temperature from 18 – 23oC (up to 5-20 tons of tubers/ha) and contains high nutrient content. Potatoes were introduced to the United States in 1719, to India in 1615, to China in 1700, to Japan in 1766 and to Vietnam in 1890. In our country, potatoes are mainly grown in the Northern provinces in Autumn season, in which the area with favorable conditions could expanded.
Origin and classification of potato crop Origins Potato (Solanum tuberosum) is native to South America. In the early 16th century, the Spanish navigators who conquered South America brought them to grow in their country. At the end of the sixteenth century, potatoes were grown in Ireland, England, Germany, France and then potatoes were grown in many other European countries. Asian countries and other continents became aware of potatoes later than European countries through the colonial policy of Europeans.
In our country, potatoes were brought to some places by the French in 1890 and mainly grown in the Red River Delta. The popular potato variety previously grown is Thuong Tin yellow-fleshed potato variety, which is derived from the Ackersegen variety, imported from France in 1890. Scientific classification 3 Table 2. Scientific classification Kingdom Plantae Order Solanales Family Solanaceae Subfamily Solanoideae Tribus Solaneae Genus Solanum Species S.
Botanical properties of the potato crop Potato plants are herbaceous perennials that grow about 60 cm high, depending on variety, with the leaves dying back after flowering, fruiting and tuber formation. Tuber stolons develop from shoots, with favorable conditions will develop into tubers, unfavorable conditions will shoot pop up to the ground to develop into branches. Potato flowers are white, pink, red, blue, or purple flowers with yellow stamens. Potatoes are mostly cross-pollinated by insects such as bumblebees, which carry pollen from other potato plants, though a substantial amount of self-fertilizing occurs as well.
Tubers form in response to decreasing day length, although this tendency has been minimized in commercial varieties. Parts of the potato plant. After flowering, potato plants produce small green fruits that resemble green cherry tomatoes, each containing about 300 seeds. Like all parts of the plant except the tubers, the fruit contain the toxic alkaloid solanine and are therefore unsuitable for consumption.
All new potato varieties are grown from seeds, also called "true potato seed" or "botanical seed" to distinguish it from seed tubers. New varieties grown from seed can be propagated vegetatively by planting tubers, pieces of tubers cut to include at least one or two eyes, or cuttings, a practice used in greenhouses for the production of healthy seed tubers. Plants propagated from tubers are clones of the parent, whereas those propagated from seed produce a range of different varieties. Biological characteristics of potato crop The growth and development stages of potato plants can be divided into 4 periods: Rest period: The dormant period of the potato means that the potato has just been harvested and is not capable of sprouting.
The factors affecting the dormancy period of potato plants depend on the variety, mechanical application and chemicals as well. After being broken dormancy, the potato tuber has the ability to sprout. Germination period: The sprouting period is the first development stage. At the nodes of the potato tubers grow small sprouts and these sprouts will develop into seedlings.
The following factors determine the germination rate of potato plants: Seed tubers: choose tubers that are not scratched, rotten, sprout fast, and healthy sprouts. Temperature: 22-30oC, if the temperature is lower, it will make it difficult for the plants to sprout Soil moisture: about 80-85%, if the soil is too dry, it will also slow down the growth of the plant, if the soil is too wet, it will cause the tubers to rot. Old tubers will sprout faster than young bulbs. If the temperature is warm, the tubers will sprout very quickly.
In one nodes tuber, the middle sprout will sprout first. The sprouts at the top of the tubers grow faster and stronger than the sprouts at the base of the tubers. On a tuber, the sprouts that grow first usually grow faster and inhibit the sprouts at the base. When one sprout is broken, other sprouts will have a chance to growth.
6 The period of tuber ray formation: After growing about 15-20 days, potato plants will form tuber rays very soon. Depending on the variety, care regime as well as the growing season, the period of tuber ray development can be short or long, usually lasting about 30- 45 days. Suitable temperature: 17-20oC. The soil must be loose, airy and clean.
The period of tuber development: This is the stage when nutrients are transported to the tuber to help the tuber grow quickly and make the tuber swell. This period lasts about 25-30 days depending on the variety. Tubers grow very quickly if the day and night temperature difference is high. Low temperature, short day light, moist soil and adequate supply of nutrients will help tubers grow very quickly.
The factors that determine the yield of potato plants: appropriate planting time, care such as watering, fertilizing. Genetics and reproductive characteristics of potatoes The diversity of potato species has been discussed in a special issue by Machida-Hirano (2015) regarding the geographical distribution and variation of species.