Screening of potato germplasm for flowering and self incompatibility (khóa luận tốt nghiệp)

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 PH.D Hanoi - 2022 COMMITMENT I hereby declare that the data and research results in this thesis are true and 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 Ph.D., 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 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 i ACKNOWLEDGEMENT ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES AND GRAPHS vi LIST OF ABBREVIATIONS vii Chapter INTRODUCTION 1.1 Introduction 1.2 Purpose 1.3 Requirement Chapter OVERVIEW OF POTATO 2.1 General introduction 2.1.1 Origin and classification of potato crop 2.1.2 Botanical properties of the potato crop 2.1.3 Biological characteristics of potato crop 2.1.4 Genetics and reproductive characteristics of potatoes 2.1.5 Some factors affect the growth and development of potato crop 13 2.1.6 Nutritional requirements of potato crop 14 2.1.7 Nutritional value 15 2.1.8 Storing potatoes 16 2.1.9 Toxicity 16 2.1.10 Role in the World Food Supply 18 2.1.11 Time to plant potatoes in Vietnam 18 2.2 The situation of potato production in the world 19 2.3 Potato production situation in Vietnam 21 2.4 Self-incompatibility gene 22 Chapter MATERIAL AND METHOD 24 3.1 Material 24 3.2 Location and time 24 3.3 Research contents 25 iii 3.4 Methods 25 3.4.1 Experimental design 25 3.4.2 Evaluation method of some agro-biological characteristics 26 3.4.3 PCR method to identify self-incompatibility genes (S11 and S16) (Olga Marcellan et al, 2012) 27 3.4.5 Evaluation of flowering 29 Chapter RESULTS AND DISCUSSION 30 4.1 Evaluation results of some agro-biological characteristics 30 4.1.1 Results of monitoring some indicators of growth and development characteristics 30 4.1.2 Results of monitoring a number of indicators on yield and quality of tubers 35 4.2 Results of PCR method to identify self-incompatibility genes (S11 and S16) 41 4.2.1 DNA extraction results 41 4.2.2 PCR results 42 4.3 Results of evaluating the flowering ability of the experimental varieties 45 Chapter CONCLUSION AND SUGGESTION 47 5.1 Conclusion 47 5.2 Suggestion 48 REFERENCES 49 iv LIST OF TABLES Table 2.1 Scientific classification Table 2.2 Representative potato genetic markers and maps 10 Table 3.1 List of screening materials 24 Table 3.2 DNA molecular markers used in the experiment 28 Table 4.1 Growth time tracking table 30 Table 4.2 Monitor stem and leaf characteristics 33 Table 4.3 Yield survey data of potato varieties 36 Table 4.4 Quality monitoring table of tubers 39 Table 4.5 PCR results to detect S11 and S16 self-incompatibility genes 43 v LIST OF FIGURES AND GRAPHS Figure 2.1 Parts of the potato plant Figure 4.1 Results of total DNA electrophoresis of 40 potato varieties 41 Figure 4.2 Electrophoresis results of PCR products detecting selfincompatibility genes S11 and S16 42 Figure 4.3 Flower images represent a number of varieties with flowering 45 Figure 4.4 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 INTRODUCTION 1.1 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 selfincompatibility 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 Ph.D., we conduct the title: "Screening of potatoes germplasm for flowering and self-incompatibility" 25 3.7 92 25.1 4644 26 2.5 50 20.0 2500 27 7.0 450 64.3 22505 28 8.8 103 11.7 5148 29 7.5 383 51.1 19163 30 5.2 160 30.8 8008 31 6.0 180 30.0 9000 32 9.8 200 20.5 10045 33 10.5 790 75.2 39480 34 12.0 410 34.2 20520 35 11.0 293 26.6 14630 36 12.0 640 53.3 31980 37 6.7 283 42.5 14238 38 6.6 270 40.9 13497 39 8.1 238 29.3 11867 40 10.4 171 16.4 8528 From the data table shows: The two varieties with the highest average number of tubers per cluster are 34 and 36 with 12 tubers/ cluster Varieties 22, 26 and 19 had the lowest average number of tubers per cluster of 2.5 and 2.8 tubers/ cluster The remaining varieties had an average number of tubers ranging from 3-9.9 tubers, only three varieties had tubers in the range of 10-11 tubers Variety number 13 had the lowest average weight of tuber (6.5 g/tuber), while variety number 33 had an average weight of tuber of 75.2 g/tuber - the highest among the varieties screening Most of the variety had an average weight of tubers ranging from 10-39 g/tube (32 varieties), only six varieties had an average tuber weight of 40-65 g/ tuber 37 Based on planting density with normal tuber size, 1m2 of land will grow clusters of potatoes (According to khuyennongvn.org.vn), hectare of land will grow 50,000 clusters, so the yield per hectare will be calculated according to formula: Yield (kg/ha) = Average number of tubers per cluster* Average weight of each tuber (kg)*50000 From the survey results, variety number 13 has the lowest yield (2015 kg/ha), variety number 33 has the highest yield (39480 kg/ha) When I compare it with the Dutch potato variety Actrice, which is a popular variety grown in some areas such as Bac Giang, Thai Binh, and Nam Dinh (GVA Joint Stock Company (Hanoi) incollaboration with the People's Committee of Dai Hoa Commune, Tan Yen District (Bac Giang), 22/12/2020) the actual yield is 16667 kg/ha (corresponding to the yield theory is 18940 kg/ha) I found that there are five varieties with higher yield than the Dutch potato variety Actrice, which are varieties 27, 29, 33, 34, 36; The tubers of the compared varieties were of similar quality 38  The following table shows the tuber quality of the potato varieties Table 4.4 Quality monitoring table of tubers Number of Color of paring Shape of tubers Color of tuber flesh Oval Light yellow Yellow Oval Light yellow Yellow Round Light yellow Yellow Long Light yellow Yellow Oval Light yellow Yellow Long Light yellow Yellow Round Light yellow Yellow Oval Light yellow Yellow Oval Light yellow Yellow 10 Oval Light yellow Yellow 11 Oval Light yellow Yellow 12 Round Light yellow Yellow 13 Round Light yellow Yellow 14 Long Light yellow Yellow 15 Long Light yellow Yellow 16 Oval Light yellow Yellow 17 Oval Light yellow Yellow 18 Oval Light yellow Yellow 19 Oval Light yellow Yellow 20 Oval Light yellow Yellow 21 Oval Light yellow Yellow 22 Oval Light yellow Yellow 23 Round Light yellow Yellow 24 Long Light yellow Yellow symbols 39 tuber 25 Oval Light yellow Yellow 26 Oval Light yellow Yellow 27 Long Light yellow Yellow 28 Oval Light yellow Yellow 29 Long Light yellow Yellow 30 Oval Light yellow Yellow 31 Round Light yellow Yellow 32 Round Light yellow Yellow 33 Oval Light yellow Yellow 34 Round Light yellow Yellow 35 Long Light yellow Yellow 36 Oval Light yellow Yellow 37 Round Light yellow Yellow 38 Oval Light yellow Yellow 39 Oval Light yellow Yellow 40 Oval Light yellow Yellow Morphological characteristics of tuber is an important criterion in research and breeding because it is directly related to the quality of commercial tubers From the survey results in Table 4.4 it can be seen that: The tuber shape of the surveyed varieties is different Varieties have round tubers including varieties, oval tubers included 23 varieties, and eight varieties have long tubers All surveyed varieties have yellow tuber paring and light yellow flesh Compared with the Dutch potato variety Actrice (GVA Joint Stock Company (Hanoi) incollaboration with the People's Committee of Dai Hoa Commune, Tan Yen District (Bac Giang), 22/12/2020), the varieties experimental have good tuber quality, especially the varieties with oval tubers 40 4.2 Results of PCR method to identify self-incompatibility genes (S11 and S16) 4.2.1 DNA extraction results Leaf samples of each variety were collected separately, all samples were young leaves, taken in the early morning, when the plants had not yet photosynthesized to avoid mixing many impurities of photosynthesis products Store in the refrigerator when it is not use immediately After obtaining the total DNA, we carried out a DNA quality check by electrophoresis in 1% agarose gel, 45 mins, 100V The results of electrophoresis (Figure 4.1) show quite clear total DNA bands to ensure the implementation of PCR reactions Figure 4.1 Results of total DNA electrophoresis of 40 potato varieties 41 4.2.2 PCR results Nested PCR assays be used with pairs of primers C1FD-C4RD and C2FC3R (Olga Marcellan et al., 2012) to detect alleles S11 and S16 The first PCR reaction is the C1FD-C4RD primer pair, then the first PCR product is used to run the second PCR reaction with the C2F-C3R primer pair After performing PCR reactions, the products were electrophoresed in 2% agarose gel of 50 V for 80 minutes We used 100bp DNA Ladder Figure 4.2 Electrophoresis results of PCR products detecting selfincompatibility genes S11 and S16 42 Through the results observed in the electrophoresis PCR products of 40 potato varieties, we found: There are wells for the line at 295bp position Thus, it can be concluded that there are varieties containing the S-RNase self-incompatibility gene with gene S11: 5, 14, 17, 25, 26, 32, 37, 39, 40 There are wells for the line at 286bp position From there, I was found that there were varieties containing the S-RNase self-incompatibility gene with the gene S16 as varieties 7, 10, 36 Table 4.5 PCR results to detect S11 and S16 self-incompatibility genes Number of symbols Variety name Gene S11 Gene S16 3-5 - - 3-4 - - 4-5 - - T1 - - 4-1 + - 3-3 - - T2 - + 5-1 - - 3-1 - - 10 T11 - + 11 2-1 - - 12 4-4 - - 13 5-4 - - 14 1-3 + - 15 5-2 - - 16 KT5-rQ - - 17 1-2 + - 43 18 2-3 - - 19 KT7 rR - - 20 5-3 - - 21 3-2 - - 22 2-4 - - 23 T3 - - 24 KT6 rR - - 25 T4 + - 26 2-2 + - 27 KT6 rh - - 28 KT1 rh - - 29 KT3 rh - - 30 KT4 rh - - 31 1-1 - - 32 1-5 + - 33 KT2 rh - - 34 T5 - - 35 1-4 - - 36 T6 - + 37 T7 + - 38 T8 - - 39 T9 + - 40 T10 + - Note: ―-‖ there has no genes, ―+‖ there has genes Among the five high yielding varieties (27, 29, 33, 34, 36), only the variety number 36 contained the S16 gene, the remaining four varieties did not contain both S11 and S16 genes 44 4.3 Results of evaluating the flowering ability of the experimental varieties Based on the actual observation results, among 40 studied varieties, some variety did not flower, some did flower but the flowers were all very small (Figure 4.3), unable to carry out cross-pollination for flowers to further evaluate the self-incompatibility genes of some varieties It is speculated that the growing conditions in the greenhouse may have affected the flowering of the potatoes Flowering varieties include the numbered varieties: 4-5, 4-1, T2, 1-3, 5-2, KT7 rR, KT6 rR, KT6 rh, KT3 rh, KT4 rh, 1-5, KT2 rh, T5, T6, T7 Figure 4.3 Flower images represent a number of varieties with flowering Five varieties with high yield are all flowering varieties 45 a) b) c) d) e) Figure 4.4 Image of tubers of high yielding potato varieties (a) Tubers of variety number 27 (KT6 rh) (b) Tubers of variety number 29 (KT3 rh) (c) Tubers of variety number 33 (KT2 rh) (d) Tubers of variety number 34 (T5) (e) Tubers of variety number 36 (T6) 46 Chapter CONCLUSION AND SUGGESTION 5.1 Conclusion From the screening results, evaluating agro-biological characteristics, the ability to contain self-incompatibility genes (S11 and S16), and the flowering ability of 40 potato varieties, we have the following conclusions: The results of the survey on agro-biological characteristics of the varieties showed that: 40 varieties gave different results in terms of growth and development characteristics, tuber quality as well as different results in yield There were experimental varieties with good quality tubers, higher yield than the control variety (Dutch potato variety Actrice), which are varieties: KT6 rh, KT3 rh, KT2 rh, T5 and T6 PCR results for detecting self-incompatibility genes showed that varieties as 4-1, 1-3, 1-2, T4, 2-2, 1-5, T7, T9, T10 contained the S11 self-incompatibility gene; The varieties T2, T11, T6 containing the S16 self-incompatibility gene are valuable resources for the selection of potato varieties by seed Observed 15 varieties capable of flowering (4-5, 4-1, T2, 1-3, 5-2, KT7 rR, KT6 rR, KT6 rh, KT3 rh, KT4 rh, 1-5, KT2 rh, T5, T6, T7) Among the five high yielding varieties (KT6 rh, KT3 rh, KT2 rh, T5, T6), only the T6 variety contained the S16 gene, the remaining four varieties did not contain both S11 and S16 genes Thus, in the breeding of hybrid potatoes, the four varieties that not contain the S11 and S16 genes can all be crossed with other varieties containing the S11 and S16 genes, but the T6 variety cannot be crossed with the varieties containing the S16 gene because of self-incompatibility, however T6 variety is still possible to cross with varieties containing the S 11 gene 47 5.2 Suggestion Continue to monitor the agro-biological parameters of 40 varieties in different growing conditions Application of DNA molecular markers to detect self-incompatibility genes in other varieties and detect other self-incompatibility genes in 40 varieties 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