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MỞ ĐẦU MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES DOAN THI HUONG GIANG RESEARCH ON IMPROVING THE SUBMERGED TOLERANCE OF R[.]

MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES DOAN THI HUONG GIANG RESEARCH ON IMPROVING THE SUBMERGED TOLERANCE OF RICE VARIETY AS996 BY MOLECULAR MARKERS Major: Genetics and Crop Breeding Code: 96.20.111 SUMMARY OF AGRICULTURE DOCTORAL THESIS Hanoi - 2022 The thesis was completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Supervisors: Prof Dr Le Huy Ham Assoc Prof Dr Luu Minh Cuc Reviewer 1: ……………………………………………… Reviewer 2: ……………………………………………… Reviewer 3: ………………………………………… …… The thesis will be defeneded in the Institute Committee of PhD Dissertation Examination at Vietnam Academy of Agricultural Sciences On /2022 The thesis can be found at: - National Library of Vietnam - Library of Vietnam Academy of Agricultural Sciences INTRODUCTION The necessary of the research Rice (Oryza sativa L.) is the most improtant food crop in Vietnam With a geographical location near the sea, having a dense system of rivers and fertile alluvial soil, wet-rice agriculture is the main job, bringing outstanding economic efficiency Growing rice plays an important role in the structure of agricultural production and plays a key role in ensuring food security, making Vietnam one of the world’s largest rice exporters Recently, due to the impact of climate change, it has caused a change in some biotic and abiotic factors in an unfavorable direction, affecting the growth and development of rice, reduce rice production in the world in general and Vietnam in particular One of the most influential factors is that waterlogging occurs in almost all rice growing areas, but the most affected area is the Mekong River Delta, where having an area and largest rice production in Vietnam (Wassmann et al., 2004; Hoanh et al., 2004) Therefore, the breeding research and development of rice sources that have improved flood tolerance and high yield are essential and meaningful in ensuring food safety and increasing farmers' incomes The study discovered that the Sub1 gene locus is the main QTL (Quantitative trait loci) controlling the quantitative trait related to the flood tolerance mechanism in rice, which has been detail mapped by Xu and Mackill, 1996 Up to now, the method of molecular marker selection and backcrossing (MABC) has been successfully applied in bringing together gene/QTL loci into new varieties AS996 rice variety is one of the high-quality rice varieties that has been popularly grown in the Mekong Delta and Southeast provinces from 2003 to present However, AS996 variety is very sensitive to waterlogged conditions, so in recent times, yield has been greatly affected when flooding conditions have increased in the field Therefore, improving the flood tolerance of rice variety AS996 is one of the urgent problems posed for the Mekong Delta Stemming from the requirements of production practice, we carried out the project: "Research on improving the submerged tolerance of rice variety AS996 by molecular markers", to create a new rice variety AS996-Sub1 capable of submerged tolerance adaptive for the Mekong Delta region Research objective Improvement of rice variety AS996 using the method of markers assisted backcrossing (MABC), to create new rice varieties AS996-Sub1 with submerged tolerance and adaptability for the Mekong Delta Scientific and practical significances 3.1 Scientific significances - The results of the thesis project will provide scientific data in gathering QTL/target gene Sub1 by MABC method applied to breeding submerged tolerance rice varieties - The research results of the thesis are also a reference for teaching, learning and scientific research, contributing to supplementing and perfecting the method of accurately assessing the presence of target genes on the expression of flood tolerance for a new rice variety with improved submerged tolerance 3.2 Practical significances - Successfully selecting and creating submerged tolerance rice variety OM351 by MABC method has opened up the possibility of wide application in the improvement of rice varieties to improve the resistance to biotic and abiotic stresses of the cultivars which are in mass production - The OM351 rice variety has agro-biological characteristics similar to AS996 but has a higher yield than the AS996 variety, especially with submerged tolerance, which will contribute to the diversity of rice varieties to be cultivated for low-lying and flooded areas in the Mekong Delta provinces Research materials and scope 4.1 Research materials Rice variety AS996 which is popular cultivated in the South provinces Rice variety IR64-Sub1 having Sub1 locus gene, this variery is imported from International Rice Research Institute (IRRI) 4.2 Research scope - The thesis focuses on crossbreeding and using molecular markers to identify individuals carrying the Sub1 locus gene and the genetic background of the AS996 variety in breeding generations - Evaluation of submerged tolerance, agro-biological characteristics, yield potential and adaptability and development of new varieties in some representative provinces of southern ecological regions The study period was from 2010 to 2017 New contributions of the thesis - Analysis of polymorphism between AS996 and IR64-Sub1 varieties identified 71 polymorphic markers Among them, 69 markers used to screen genetic background of the ASS996, the two markers ART5 and SC3 are located in the Sub1 locus gene to select individuals carrying the Sub1 locus in the backcross populations - Applying the MABC method has successfully improved, selected and created the submergence rice variety AS996-Sub1 (newly named OM351) The OM351 vaeiety has agro-biological characteristics, quality and resistance to pathogen and diseases similar to AS996, higher yield than AS996, especially with 14-days submerged tolerance ability Thesis structure The thesis consists of 154 pages including parts: Introduction (4 pages); Chapter Literature Review (36 pages); Chapter 2: Materials, Contents and Research Methds (20 pages); Chapter 3: Research Results and Discussion (72 pages); Conclusions and Recommendations (2 pages); List of published works related to the thesis (1 page); References (19 pages) included 25 Vietnamese documents, 121 English documents and websites The thesis has 41 data tables, 32 figures, appendices and published works Chapter LITERATURE REVIEW 1.1 Impact of climate change to agricultural production According to the assessment of the Intergovernmental Panel on Climate Change (IPCC), Vietnam is one of the five countries most affected by climate change In the climate change scenario to the year 2100: 1,8 million hectares of rice land are affected by climate change; accounting for 91,8% of the total area of rice land in the whole region; 1,8 times higher than the climate change scenario in 2030; increased 2,2 times compared to 2020 Accordingly, the rice cultivation area lost 2,2 million hectares and the damaged rice output corresponds to 55,6% of the total rice production of the region The most affected rice cultivation areas are the key ricegrowing provinces in the Dong Thap Muoi region, the Long Xuyen Quadrangle such as Kien Giang, Long An, An Giang, and Dong Thap The provinces that lost the most rice production were Kien Giang, Soc Trang, and Tra Vinh (Huong and Pathirana, 2013) 1.2 Study on the mechanism of submerged tolerance of rice Under flooded conditions, the morphological changes in the roots and shoots were affected, the factors involved in the plant growth were affected, such as number of branches, plant height, relative growth rate, internode length, chlorophyll a, b When flooding happened, the oxygen content of the water falls below the level suitable for plant growth, respiration changes from aerobic to anaerobic causing an energy crisis It is the anaerobic fermentation that leads to the accumulation of cytotoxic compounds In the condition of complete flooding, the plant's organs suddenly reduce the rate of gas diffusion, limiting the introgression of oxygen and thus photosynthesis is also reduced, the process of respiration increases rapidly, taking up all the stored carbohydrates, leading to plant death (Das et al., 2009) The higher photosynthetic capacity in flood-tolerant cultivars is due to the protection of photosynthetic organs, such as higher chlorophyll content, better stomatal conductivity after flooding (Panda and Sarkar, 2017) The good flood tolerance mechanism is also related to the accumulation of unstructured carbohydrates The unstructured carbohydrate composition is necessary for the survival of the plant under adverse conditions Carbohydrate concentration before flooding is considered a key trait for survival under flooded conditions (Panda and Sarkar, 2014) The carbohydrate stored in the plant is used to provide the energy needed to sustain the plant while submerged Research groups have shown that flooded varieties contain 30%-50% of carbohydrate reserves (Afrin et al., 2018) Plant hormone components such as ethylene gas, gibberellic acid, and abscisic acid are considered to play important roles through synergistic and antagonistic actions for plant survival under flooded conditions (Huang et al., 2019) ) The activities of ethylene and gibberellic acid stimulate prolonged plant growth under flooded conditions (Voesenek et al., 2013; Goswami et al., 2017) Ethylene increases the response of growth tissues to gibberellic acid by reducing abscisic acid, a potent antagonist of gibberellic acid, which enhances the plant's tolerance to flooding (Du et al., 2014; Locke et al., 2018 ) Experimental inhibition of gibberellin biosynthesis before flooding increased the survival rate of plants that were flooded many times Plant hydrogen peroxide (H2O2) plays a key role in flooding, as a signaling molecule that regulates various physiological processes (Fukao et al., 2019) This molecule reduces inhibitory levels in low light, and can alter expression levels of hundreds of genes in plants (Yun et al., 2010) Peroxidase activity in rice leaf sheaths increased from preinundation to post-flood in all tested varieties This increase was stronger in tolerant varieties The molecular mechanism of "submergence" is controlled by the Sub1A gene, formed in response to elevated ethylene levels, which accumulate during flooding, to inhibit both ethylene production and gibberellic responsiveness acid (GA), leading to a long-lasting inhibition during submersion The decreased GA responsiveness is due to the accumulation of Della proteins such as slender rice (SLR1), which inhibits GA responses (Fukao and Bailey-Serres, 2008) The Sub1A1 gene increases brassinosteroid (BR) content after flooding by downregulating the transcription of the BR fusion gene, and this causes GA repression by metabolism of the GA-inactivating enzyme OsGA2ox7 (Schmitz et al., 2013) Furthermore, recent work has shown that Sub1A suppresses carbohydrate and nitrogen metabolism and controls responses to leaf growth-regulating hormones under flood stress (Alpuerto et al., 2022) Tolerance to full submergence is a genetic trait that helps rice plants to recover from full submersion (10-14 days) 1.3 The method of selection by molecular markers combines backcrossing and its application in plant breeding DNA molecular markers are markers that are polymorphic in nature In this study, the SSR (Simple Sequence Repeats) indicator is used, which amplifies the simple repeats or microsatellites Microsatellites are sequences of DNA that repeat in an orderly fashion, consisting of repeating units from to nucleotides, in a short repeating pattern and several dozen times The advantage of the SSR directive is that it is easy to implement and less expensive SSR is a co-dominant marker capable of detecting very high polymorphisms and is species-specific SSR markers are accurate and effective in studying genetic diversity, in analyzing organism genomes, building genetic maps of gene linkages, and selecting for resistance/tolerance to some biological and abiotic traits, learning to apply in breeding The method of selective breeding by molecular markers combined backcross (MABC) has been widely used in recent years and has brought many successes in plant breeding The aim of MABC is to transfer one or several genes/QTLs of interest from a genetic material source into elite lines/varieties to improve the target trait (Neeraja et al., 2007), MABC applied based on DNA markers that have a relationship or association with the gene/QTL of interest instead of just phenotyping the target trait like traditional backcross methods Recipient plant genomes can rapidly combine target gene selection and combined genetic background selection using traditional backcross methods (Hasan et al., 2015) The MABC breeding method is very important, supporting traditional selection and determining the success of the breeding process (Collard et al., 2005) There have been many researches on applying the method of using gene-linked molecular markers in plant breeding, and achieved success in the world and in Vietnam 1.4 Studies on the application of molecular markers in the selection and breeding of submerged tolerant rice varieties The International Rice Research Institute mapped the QTL associated with the flood tolerance trait (Sub1) located on chromosome 9, and successfully introduced rice varieties for flooded areas by MABC breeding The research team at IRRI has selected and released several QTL/Sub1 gene carriers in countries including varieties in Indonesia, varieties in Nepal, variety in Burma, varieties in India and varieties in Bangladesh and varieties in the Philippines In Indonesia, in two years, Sub1 gene was successfully transferred into two rice varieties Ceherang and PSB RC18 by MABC method (Septiningsih et al., 2015) In Vietnam, Dao Van Khoi has applied a molecular marker to breed flood-tolerant SHPT2 with Sub1 gene from Khang Dan 18 rice variety, which has been recognized and put into production in Vietnam (Dao Van Khoi, 2019) Most of the other results have only been recorded at the initial level of success in crossing over and checking the presence of the Sub1 gene locus in lines, creating breeding materials or promising lines (Ta Hong Linh, 2012; Cuc et al 2012, Luu Thi Ngoc Huyen et al., 2014) In summary: MABC breeding method is currently one of the most effective methods in improving the breed with the desired trait through the integration of target genes into the genome The development of broad-adaptive rice varieties that respond to changes in biotic and abiotic stresses increased by climate change is needed Researches on improving rice varieties in the world as well as in the country are always aimed at that In fact, in recent years, flooding is one of the leading and most serious challenges facing of the rice production industry in Vietnam Therefore, improving and enhancing the submerged tolerance of popular rice varieties outside of production in the southern provinces is one of the issues that need to be prioritized today Chapter RESEARCH MATERIAL, CONTENTS AND METHODS 2.1 Research Materials The rice variety AS996, receiving the tolerance gene, is widely grown in production in the southern provinces The rice variety IR64-Sub1, carrying the Sub1 submergence locus gene, was imported from the International Rice Research Institute (IRRI) The use of 400 SSR molecular markers, as well as agricultural materials and specialized materials in molecular biology 2.2 Research content Content Evaluation of starting materials for the improvement of submerged tolerance rice varieties Content Crossbreeding and selection of crossbred individuals carrying both the Sub1 - submerged tolerance locus gene and the AS996 genetic background by molecular markers and backcrossing (MABC) Content Evaluation of submergence tolerance, agro-biological characteristics and yield potential of some promising rice lines/varieties in selective generations Content Production test and author’s test of submerged tolerance rice variety AS996-Sub1 (OM351) in some southern provinces 2.3 Research Methods 2.3.1 Evaluation of starting materials for the improvement of submergent tolerant rice varieties 2.3.1.1 Material source evaluation * The experiment to evaluate the materials was conducted with rice varieties a Experimental design: The experiment was arranged in a randomized complete block (RCB) with replications Area of each experimental plot: (5 m x m) = 10 m2 Total experimental area: 10m2/plot x number of test cells x replicates (excluding protection band) Implementation period: Fall-Winter 2010 b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length); Yield components (number of branchs/clump, number of firm seeds/branch, weight of 1000 seeds, percentage of empty seeds, actual yield) * The experiment to evaluate the tolerance of materials was carried out with rice varieties according to the method of Pamplona et al., 2007 a Experimental design: a completely randomized one-factor experiment with three replicates, 24 seeds/replication Conduct the assessment when the plant has developed a new leaf after 15-21 days of submergence, count the number of live plants b Calculating submergent tolerance according to IRRI's scale (2013) The rice varieties participating in the experiment were evaluated according to the following formula: CS%= (S1%/S2%)*100 In which: CS% – survival rate; S1% – survival rate of the variety to be evaluated; S2% – survival rate of the control variety carrying the Sub1 gene Scale for assessing submerged tolerance based on CS%: score (CS% = 100 or more); point (CS% = 95-99%); score (CS% = 75-94%); score (CS% = 5074%); score (CS% = 0-49%) 2.3.1.2 Evaluation of broodstock in the selection and breeding of submergent tolerant rice varieties a Experimental design: The experiment was arranged in a randomized complete block (RCB) with replications Area of each experimental plot: (5 m x m) = 10 m2 Total experimental area: 10m2/plot x number of test cells x replicates (excluding protection band) Implementation period: Winter-Spring 2010-2011 b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length); Yield components (number of branchs/m2, number of firm seeds/branch, weight of 1000 seeds, actual yield) 2.3.1.3 Determination of molecular marker on 12 chromosomes for polymorphisms between IR64-Sub1 and AS996 a DNA extraction and purification techniques Rice DNA was extracted and purified by CTAB method (Laboratory of Genetics, University of Ghent, Belgium) b Survey of polymorphism between the two parent breeds A total of 400 SSR markers on 12 chromosomes were selected *PCR with SSR primers were performed in 0.2 ml eppendorf or 96-position PCR plate and amlified on an Eppendorf Master Cycle Pro S machine * Electrophoresis analysis of PCR results on polyacrylamide gel: * Recording results: for each SSR marker, if the DNA band size is different between the surveyed varieties, it will be recorded as having polymorphism and will be used for screening for resistance genes or genetic background in the folowing steps 2.3.2 Crossbreeding and selection of crossbred individuals carrying both the Sub1 submerged tolerance locus gene and the AS996 genetic background by molecular markers and backcrossing (MABC) 2.3.2.1 Method of sexual hybridization, back-crossing Conduct crossbreeding between variety AS996 (mother) with variety having Sub1 tolerance locus IR64-Sub1 (father) Using hybrid generations F1, BC1F1, BC2F1 backcrossed with AS996 to create hybrid population BC1F1, BC2F1, BC3F1 respectively 2.3.2.2 Selection of crossbred individuals carrying the Sub1 locus gene and the genetic background of AS996 using SSR molecular markers Sowing F1 hybrid seeds of generations BC1F1, BC2F1, BC3F1 The F1 hybrid seeds were grown until 20 days old, then DNA extraction was carried out, the presence of Sub1 locus gene was checked, and the genetic background of AS996 received by polymorphic molecular markers that were found in previous step Select individuals having Sub1 locus gene and largest genetic background of AS996 in each generation aimed to create BC1F1, BC2F1, BC3F1 populations Experimental steps were carried out as in section 2.3.1.3 Data were recorded and processed on Graphical genotypes software (GGT2.0) and other statistical analysis methods The regulation was that, the DNA band of IR64-Sub1 variety was A, the DNA band of AS996 variety was B, carrying both bands in the heterozygous state was H 2.3.3 Evaluation of submergence tolerance, agro-biological characteristics and yield potential of some promising rice lines/varieties in selective generations 2.3.3.1 Evaluation of submerged tolerance of some rice lines/varieties in BC3F3 breeding generation - Using the method of Pamplona et al., 2007 (mentioned in section 2.3.1.1) to evaluate When the plants get more leaf during the 15 day recovery period, on the 21st day, evaluate the results by counting the number of live plants and calculate the survival rate according to the formula as in section 2.3.1.1 2.3.3.2 Evaluation of some agro-biological characteristics and yield potential of some promising rice lines/varieties from BC3F3 to BC3F5 generations a Experimental design: The experiment was arranged in a randomized complete block (RCB) with replications Area of each experimental plot: (5 m x m) = 10 m2 Total experimental area: 10 m2/plot x number of test plots x replicates (excluding guard area) Implementation period: Fall-Winter 2013; Winter Spring 2013-2014 and Summer Fall 2014 b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length); Level of infection with pathogens and diseases; Yield components (number of branchs/clump, number of firm seeds/branch, weight of 1000 seeds, percentage of empty seeds, actual yield) c Cultivation technique: - The soil is carefully made by machine, picking up weeds, leveling, ensuring there is no stagnant water, creating trenches around to drain - Sowing technique: planting density of 40 clumps/ m2 - Fertilizer method: + Primer: 100% organic fertilizer + 100% phosphorus + 25% urea + Promote: Phase 1: 7-10 days after sowing: apply 40% urea Phase 2: After phase one 10 days: apply 20% urea + 40% potassium Phase 3: When the rice is 1-2 cm long, apply 15% urea + 60% potassium - Pest control: applied according to the scale of technical regulation QCVN0155:2011/BNNPTNT (National regulation on testing the value of cultivation and use of rice varieties) d Method of traits evaluation: according to "National technical regulation on testing the value of cultivation and use of rice varieties" (QCVN 0155:2011/BNNPTNT)" 2.3.3.3 Evaluation of submerged tolerance, comparison of rice varieties under normal and submerged conditions * Under normal conditions: a Experimental design: The experiment was arranged in a randomized complete block (RCB) with replications Area of each experimental plot: (5 m x m) = 10 m2 Total experimental area: 10 m2/plot x number of test cells x replicates (excluding protection area) Implementation period: Winter-Spring 2014-2015; Summer Fall 2015 and Winter Spring 2015-2016 b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length, arista neck release, plant stiffness, leaf canopy); Level of + Evaluation of material sources, artificial submerged tolerance, agro-biological characteristics of submerged tolerance rice lines/varieties in green house system, fields belong to the Mekong Delta Rice Institute (Tan Thanh, Thoi Lai, Can Tho) + Testing submerged tolerance rice varieties in the national testing system in the provinces representing ecological regions: Mekong Delta (Tien Giang, An Giang, Co Do, Thoi Lai, Kien Giang), Southeast region (Binh Thuan, Tay Ninh) + Authorization trial of submerged tolerance rice varieties AS996-Sub1 (OM351) tested in mass production in Dong Thap, An Giang, Tra Vinh, Bac Lieu, Long An, and Ben Tre provinces Chapter STUDY RESULTS AND DISCUSSION 3.1 Evaluation of starting materials for the improvement of submerged tolerance rice varieties 3.1.1 Evaluation of material resources, selection of parent varieties 3.1.1.1 Evaluation of material resources The experiment was conducted on varieties, they were INPARA3, IR5S713-2B-8-2B-1-2, IR64-Sub1, BR11-Sub1, PSB-Re68-Sub1, Samba MahsuriSub1, AS996 and IR42 The experimental results shown that: the variety with short growing time, high yield in the imported variety group, with the highest submergence tolerant among the tested lines/varieties is IR64-Sub1 (yield 57,1 quintal/ha) Combined with the results of the assessment of submergence tolerant, the variety IR64-Sub1 has the highest submergence tolerant among the tested rice lines/varieties (point 1) with 100% survival rate after the experiment, play the standard tolerance role, and at 78,9% compared to itself The nature of IR64-Sub1 is an IR64 variety that carries the QTL/ submerged tolerance gene (Sub1) which is currently being grown quite popularly in some South and Southeast Asian countries, including Vietnam IR64-Sub1 variety has good genetic background, wide adaptability and stable yield over the years, especially has the best submerged tolerance compared to the imported variety that has been tested in artificial conditions It is the most suitable variety to use as the donor for Sub1 tolerance gene The rice variety AS996 is not submerged tolerance, but is suitable in the Mekong Delta region, giving the highest yield (58.3 quintals/ha) of the surveyed varieties, suitable for use as the recipient Sub1 tolerant gene in the study for breeding rice varieties that are submerged tolerance 3.1.1.2 Results of evaluation of parental materials for breeding purpose After determining the source of materials to use as donor variety (IR64-Sub1) and recipient variety (AS996) for submerged tolerance gene Sub1, evaluate and survey both varieties for agro-biological characteristics and productivity factor again The results of the survey and evaluation shown that: Regarding the growth time: the two parent varieties are in group A1 (short days), ranging from 102-110 days in the Autumn-Winter season and 98-103 days in the Winter-Spring season About tree height: variety IR64-Sub1 has a tree height (98.5cm) lower than that of AS996 (105.3cm) About arista length: variety IR64-Sub1 has a shorter arista length (20.9cm) than that of AS996 (21.7cm) Rice variety AS996 has a larger number of firm seeds per arista (103.8 seeds) than IR64-Sub1 (98.7 seeds) The number of flowers/m2 of the two varieties were similar Yields in both Autumn-Winter and Winter-Spring seasons of AS996 variety were higher than IR64-Sub1 Thus, through the above survey and evaluation step, once again, it was confirmed that the AS996 variety was decided to be used as a recipient of the submerged tolerance gene and the IR64-Sub1 variety was used as a donor for the submerged gene is appropriate in this study 3.1.2 Results of detemination of markers on 12 chromosomes for polymorphisms between two varieties IR64-Sub1 and AS996 as parents in hybrid populations A total of 400 markers on 12 chromosomes were screened to find polymorphic markers between AS996 and IR64-Sub1 Figure 3.3: Result of testing some SSR markers to find polymorphic markers between ASS996 and IR64-Sub1 Note: the upper line is the name of the SSR markers used, the lower line if the symbol P1: AS996, P2: IR64-Sub1, standard 25bp ladder The percentage of polymorphic markers were summarized in Table 3.19 shown that: A total of 71/400 markers for polymorphisms, accounting for 17.75% of the markers were screened On chromosome one, 32 markers were screened, found markers for polymorphism, accounting for 18.75% On chromosome two, screening 31 markers found markers for polymorphism, accounting for 19.03% On chromosome three, screening of 40 markers found markers for polymorphism, accounting for 10% On chromosome four, screening 33 markers found markers for polymorphism, accounting for 18.18% On chromosome five, screening 27 markers found markers for polymorphism, accounting for 18.52% On chromosome six, screening 23 markers found markers for polymorphism, accounting for 21.74% On chromosome seven, screening 31 markers found markers for polymorphism, accounting for 16.13% On chromosome eight, screening 23 markers found markers for polymorphism, accounting for 17.39% On chromosome nine, screening 67 markers found 12 markers for polymorphism, accounting for 17.91 % On chromosome ten, screening 33 markers found markers for polymorphism, accounting for 15.15 % On chromosome eleven, screening 30 markers found markers for polymorphism, accounting for 16.67% On chromosome twelve12, screening of 30 markers found markers for polymorphism, accounting for 16.67% Generally, on chromosomes, the rate of markers for polymorphism is highest on chromosome two, and the rate of polymorphism is lowest on chromosome three Table 3.19 Ratio of polymorphic markers on 12 chromosomes No of screened No of polymorphic Ratio of Chro markers markers polymorphic (%) 32 18.75 31 29.03 40 10.00 33 18.18 27 18.52 23 21.74 31 16.13 23 17.39 67 12 17.91 10 33 15.15 11 30 16.67 12 30 16.67 Total 400 71 17.75 Molecular markers for polymorphisms between AS996 and IR64-Sub1 as parents in breeding hybrid populations were used to screen the genetic background of the progeny in backcross populations Two markers associated with the QTL region/Sub1 gene and located on either side of the Sub1 locus are ART5 (6.3Mb) and SC3 (6.6Mb), found polymorphic between the two cultivars, IR64 -Sub1 and AS996, were used to select individuals carrying the Sub1 target gene in backcross populations and in breeding generations 3.2 Results of crossbreeding and selection of crossbred individuals carrying both the Sub1 - submerged tolerance locus gene and the AS996 genetic background by molecular markers and backcrossing (MABC) 3.2.1 Results of genotyping and selection of individuals carrying Sub1 loci and genetic background of AS996 in the F1 and BC1F1 population To identify individuals carrying the Sub1 target gene locus in the BC1F1 hybrid population, two markers SC3 and ART5 that are closely associated with the Sub1 target gene locus, were used Out of 120 individuals in the BC1F1 population, 56 individuals carrying the Sub1 gene locus were identified, having heterozygous genotype (H) with the two markers used for screening, ART5 and SC3 Continuing to use 69 polymorphic markers on 12 chromosomes to screen the genetic background of 56 selected individuals, putting the data into analysis in GGT v.2.0 software The results showed that the software calculated the gene background value of AS996 in the whole population, the average rate was 75.5% (carrying the A allele) and the average number of individuals carrying the heterozygous gene was 24.5% (carrying the H allele) Among BC1F1 population, individual number 16 was selected with a background rate of the AS996 variety of 80% (A allele) and a heterozygous genotype rate of 20% (H allele) to develop the next population, can be backcrossed in creating BC2F1 generation for breeding Individual number 16 was selected to cross back with the AS996 cultivar to develop BC2F1 population 3.2.2 Results of selecting individuals carrying Sub1 locus for submergence tolerance and genetic background similar to AS996 in BC2F1 population The BC2F1 generation was made up of 128 individuals Extracting DNA of BC2F1 individuals and conduct genotyping of individuals at the tillering stage Results of analysis of 128 BC2F1 individuals with ART5 and SC3 obtained 62 individuals carrying the Sub1 gene locus, expressed as heterozygous for both these markers Continue to select individuals that both carry the target gene locut Sub1, and carry the closest genetic background to the AS996 rice variety All 62 BC2F1 individuals carrying the Sub1 target gene locus were further genotyped with 69 polymorphic markers on 12 chromosomes The results of genetic background assessment data were processed on GGT v.2.0 software to select individuals with the closest genetic background to the AS996 cultivar The software calculated the average genetic background value of AS996 in the population was 88.1% (carrying allele A) and the average number of individuals carrying the heterozygous gene was 11.9% (carrying allele H) ) Among BC2F1 population, individual number was selected with 93.8% AS996 genetic background (A allele), and 6.2% heterozygous genotype (H allele) to develop the backcross population in creating the BC3F1 generation for breeding 3.2.3 Results of selecting individuals carrying Sub1 locus for submergence tolerance and genetic background similar to AS996 in BC3F1 population From individual BC2F1 number 5, backcross with rice variety AS996 to create 132 individuals of BC3F1 generation population Conduct genotyping of individuals at the tillering stage of the tree, genotype analysis of individuals in BC3F1 population with two markers linking the Sub1 locus, ART5 and SC3 The results obtained 58 individuals carrying the Sub1 locus, expressed as heterozygous for both these markers Similar to BC1F1 and BC2F1 populations, continue to select individuals that both carry the Sub1 target gene locus, and carry the closest genetic background to the AS996 variety All 58 individuals in the BC3F1 population carrying the Sub1 locus were further genotyped with 69 polymorphic markers on 12 chromosomes The genetic background assessment results were processed on GGT v.2.0 to select individuals with the closest genetic background to AS996 The software calculated the genetic background value of the AS996 variety in the whole population as 93.9% (carrying the A allele), and the number of individuals carrying the heterozygous gene (carrying the H allele) reached the rate of 6.1% In the BC3F1 population, individual number 56 was selected with 98.9% genetic background closest to AS996 (A allele), and 1.1% heterozygous genotype (H allele) for development breeding populations 3.2.4 Results of selecting individuals carrying the Sub1 locus for submergence tolerance at the homozygous stage From individual number 56 in BC3F1 population, self-pollination to create the next populations In the BC3F2 generation, two Sub1-loci-linked markers, ART5 and SC3, continued to be used to identify homozygous carriers of the Sub1 locus The analysis results showed that individuals 1, 2, 3, 4, 5, 6, 7, 10, 13, 18, 19 carried the homozygous Sub1 locus at the position of the SC3 marker Individuals 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 15, 16, 18, 19, 20, 21 carried the homozygous Sub1 locus at the ART5 position Combining the results of homozygous genotype analysis of both ART5 and SC3 markers, BC3F2 individuals number 1, 2, 3, 4, 6, 7, 10, 13, 18, 19 carrying Sub1 loci for both screening markers They were selected to develop BC3F3 populations Further testing the presence of the Sub1 locus by two markers, SC3 and ART5 in the BC3F3 population The results showed that all BC3F3 individuals carried the Sub1 locus homozygous So far, it has been shown that the successful integration of the Sub1 submerged tolerance locus into the AS996 rice variety This study has contributed to the successful application of MABC technology in plant breeding in Vietnam 3.3 Results of evaluation submergence tolerance, agro-biological characteristics and yield potential of some promising rice lines/varieties in selective generations 3.3.1 Results of assessing submerged tolerance of some rice lines/varieties in BC3F3 breeding generation Along with the experiment to evaluate some agro-biological characteristics, the lines C1; C2; C3; C4, C6; C7; C10, C13, C18, C19 bearing the homozygous Sub1 locus in the BC3F2 generation were evaluated for their tolerance to submergence in artificial conditions Table 3.20: Results of assessing submerged tolerance of experimental lines carrying gene locut Sub1 Lines/ having Survival Assessing No Score Sub1 varieties ratio (%) submerged tolerance C1 68,3 Medium tolerance + C2 + 88,3 Good tolerance C3 73,3 Medium tolerance + C4 73,3 Medium tolerance + C6 76,7 Medium tolerance + C7 + 88,3 Good tolerance C10 + 86,3 Good tolerance C13 + 91,7 Good tolerance C18 65,0 Medium tolerance + C19 61,7 Medium tolerance 10 + 16,7 No tolerance 11 AS996 86,7 12 IR64-Sub1 + Good tolerance 0,0 No tolerance 13 IR42 (Đ/c) Note: (+): having Sub1; (-): don’t have Sub1; Đ/c: control sensitive cultivar The results of the evaluation in table 3.20 shown that: The group of lines/varieties with good submerged tolerance (point 1) includes C2; C7; C10; C13 and IR64-Sub1 with the survival rate of plants after 14 days of complete submergence was 88.3%; 86.3% ; 91.7% and 86.7% These lines all have submerged tolerance (point 1) equivalent to the donor cultivars IR64-Sub1, and are also play the submerged tolerant control The variety IR42 is a sensitive control variety that cannot tolerate flooding After 13 days of submergence, 100% of the experimental plants were completely dead (point 9) The remaining lines in the experiment are all moderately flooded (score 7) 3.3.2 Evaluation of agro-biological characteristics and yield potential of some rice lines/varieties in breeding generations In parallel with the assessment of submerged tolerance, growth time, yield and some yield components, in the Autumn-Winter season 2013, 10 lines of BC3F3 having the Sub1 locus were further evaluated to select the preferred lines Table 3.21: Evaluation results of lines carrying the Sub1 locus at generation BC3F3 in Autumn-Winter 2013 at Mekong Delta Rice Institute No Increase / decrease Growth Arista Firm seed Yield Lines/ Plant P.1000 compare time /m2 /arista (quinta/ varieties heigh (cm) (gam) to the (day) (arista) (seed) ha) control cultivar (%) C1 C2 C3 C4 C6 106 109 107 107 108 101,0 111,3 103,7 114,7 102,3 285,2 253,8 281,4 282,6 288,3 98,9 100,9 108,6 98,2 105,2 26,6 26,1 25,9 25,9 25,8 4,7 4,9 4,9 4,9 5,0 4,4 8,9 8,9 8,9 11,1 C7 C10 C13 C18 102 103 102 107 105,0 103,0 104,3 114,0 262,2 273,6 299,6 261,3 82,9 94,9 114,0 98,0 26,4 25,7 26,8 23,3 5,1 5,1 5,5 4,2 13,3 13,3 22,2 -6,7 10 C19 109 113,0 264,7 118,3 21,5 4,3 -4,4 AS996 (Đ/c) 12 IR64-Sub1 13 IR42 11 103 105,0 267,3 87,4 25,6 4,5 - 106 105 103,0 91,7 321,6 303,0 80,9 73,0 26,5 25,4 4,2 3,5 -6,7 -22,2 CV% 6,6 7,5 13,0 1,7 7,9 LSD0,05 11,8 35,5 21,3 0,7 0,6 Thus, the evaluation results of some agro-biological characteristics, yield and tolerance to submergence in artificial conditions of the experimental lines showed that: lines C7, C10 and C13 had the same growth time as control AS996, on the other hand, these three lines had good submerged tolerance (point 1), yielding 13.3% to 22.2% higher than the control So three lines C7; C10 and C13 were selected to develop the BC3F4 generation Continue to evaluate the agro-biological characteristics of this population and select promising lines The results are shown in Table 3.22 Table 3.22: Evaluation results of lines carrying the Sub1 locus at generation BC3F4 in Winter-Spring 2013 at Mekong Delta Rice Institute No Firm Growth Plant Arista Lines/ seed time heigh /m varieties /arista (day) (cm) (arista) (seed) C7-1 C10-1 C13-1 101 101 99 108,0 109,3 101,7 364,3 398,7 457,6 C13-4 101 110,3 410,7 C13-3 98 112,7 366 C13-2 C13-5 C13-6 AS996(Đ/c) 10 IR64-Sub1 CV% 104 99 101 101 114,7 109,3 103,3 100,7 386,7 377,3 384,3 402,3 Increase/ Ratio decrease P.1000 Yield* empty seed (quinta/ compare to seed (gam) ha) the control (%) cultivar (%) 117,2 10,5 120,4 11,1 130,6 8,9 27,2 27 27,4 5,9 b 4,6 6,0 b 5,1 6,8 a 19,4 b 3,5 1,4 111,3 10,6 27,2 5,9 108,2 27,2 5,7b 9,3 114,2 12,6 119,5 12,2 105,3 9,6 103,7 11,9 b 27,5 5,81 27,5 5,8 b 2,8 5,9 b 4,8 5,7 b 0,0 b -0,3 27,6 27,3 98 90 394,7 99,6 12 26,5 5,6 - 4,7 8,7 11,9 8,5 1,5 6,5 2,5 LSD0,05 8,5 58,6 23,1 1,6 0,7 0,7 *Note: Same letters in the same column indicate no significant difference, different letters indicate significant difference Through the experiment to evaluate the growth time and yield of the experimental lines, the promising line C13-1 with the shortest growing time and the highest yield was selected The C13-1 line was further evaluated and selected in the Summer- Autumn 2014 when developing the BC3F5 population From the C13-1 line continued to develop into BC3F5 population Through the evaluation of phenotype and agro-biological characteristics and yield potential, promising lines were selected The results are evaluated in Table 3.23 Table 3.23: Evaluation results of lines carrying the Sub1 locus in the BC3F5 generation in the Summer-Autumn 2014 at the Mekong Delta Rice Institute No 10 Lines/ varieties C13-1-1 C13-1-3 C13-1-4 C13-1-5 C13-1-6 C13-1-7 C13-1-8 C13-1-9 AS996(Đ/c) IR64-Sub1 Growth time (day) 100 96 101 105 100 105 105 102 102 105 Increase/ decrease Firm Ratio Plant Arista P.1000 Yield* compare seed empty seed (quinta/ to the heigh /m2 /arista seed (gam) control ha) (cm) (arista) (seed) (%) cultivar (%) 102 110 105,3 115,7 103,3 112,3 104,7 114 106 291,9 268,9 344,3 289,3 295 260,5 288,1 280,3 305,0 98,2 85,5 108,3 93,7 102,8 98,5 102,4 92,5 90,9 11 9,9 6,6 10,6 9,8 11,1 11 9,5 10,4 27,2 27 27,4 26,5 26,4 26,7 26,5 26,3 27,5 59,3b 5,7 59,6 b 6,2 66,0 a 17,6 58,1 b 3,6 57,5 b 2,5 58,1 b 3,6 58,3 b 3,9 59,4 b 5,9 56,1 b 0,0 b 0,7 104 328,3 92,6 10,5 26,7 56,5 CV % 6,8 8,9 8,8 11,3 1,7 5,7 LSD0,05 12,5 45,0 14,6 1,9 0,8 0,6 *Note: Same letters in the same column indicate no significant difference, different letters indicate significant difference Conduct selection in promising lines C13-1-1, C13-1-3, C13-1-4, C13-1-5, C131-6, C13-1-7, C13-1-8 and C13-1-9 in BC3F5 generation with selection objectives: short growth time (shorter than 100 days), high number of arrista/ m2, low percentage of empty seeds and actual yield of over 60 quintals /ha In which, the priority growth time with factor and the actual yield with factor 10 The results of line selection by Nguyen Dinh Hien's Ver 1.0 line selection software (Appendix 3) have selected line (C13-1-4) meets the above criteria Line C13-1-4 carries the following characteristics: growth time (100 days); plant height (105.3 cm); number of arista/m2 (344,3 aristas); number of firm seeds/arista (108.3 seeds); low percentage of empty seeds (6.6%); P.1000 seeds (27.4 grams) and actual yield reached 66.0 quintals/ha Thus, the C13-1-4 line was selected to develop the BC3F6 generation in the Autumn-Winter 2014 and was named as AS996-Sub1